HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency and immune escape. The presence of auxiliary proteins-encoding ORFs in HTLV-3, the latest HTLV to be discovered is unknown. STLV-3 viruses are almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using RT-PCR, we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8 and 9 kDa, and confirmed the presence of the previously reported RorfII transcript. The existence of those viral mRNAs was confirmed using splice site-specific RT-PCR in ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in calreticulin signal, similar to HTLV-1 p12. Despite that p8, RorfII and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal, only p8 is found in the nucleolus. p8 location in the nucleolus is linked to a bipartite NoLS. p8, and to a lesser extent p9, repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax3 and APH-3 promote cellular transformation. Altogether, these results complete the characterization of the newly described PTLV-3 virus.
HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-?B expression, and HIV-1- and NF-?B-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of I?B-?, with increased accumulation of cytoplasmic NF-?B. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-?B-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection.
Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-?B in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.
The prefrontal cortex (PFC) is dysregulated in neuroAIDS and during cocaine abuse. Repeated cocaine treatment upregulates voltage gated L-type Ca(2+) channels in pyramidal neurons within the rat medial PFC (mPFC). L-type Ca(2+) channels are also upregulated by the HIV-1 neurotoxic protein, Tat, but the role of Tat in pyramidal cell function is unknown. This represents a major knowledge gap as PFC pyramidal neurons are important mediators of behaviors that are disrupted in neuroAIDS and by chronic cocaine exposure. To determine if L-channel-mediated Ca(2+) dysregulation in mPFC pyramidal neurons are a common neuropathogenic site for Tat and chronic cocaine, we evaluated the electrophysiological effects of recombinant Tat on these neurons in forebrain slices taken from rats 1-3 days after five, once-daily treatments of cocaine (15 mg/kg, ip) or saline. In saline-treated rats, bath-applied Tat facilitated membrane depolarization and firing. Ca(2+) influx was increased (indicated by prolonged Ca(2+) spikes) with low concentrations of Tat (10-40nM), but reduced by higher concentrations (80-160nM), the latter likely reflecting dysfunction associated with excessive excitation. Tat-mediated effects were detected during NMDA/AMPA receptor blockade, and abolished by blocking activated L-channels with diltiazem. In neurons from cocaine-treated rats, the Tat-induced effects on evoked firing and Ca(2+) spikes were significantly enhanced above that obtained with Tat in slices from saline-treated rats. Thus, glutamatergic receptor-independent over-activation of L-channels contributed to the Tat-induced hyper-reactivity of mPFC pyramidal neurons to excitatory stimuli, which was exacerbated in rats repeatedly exposed to cocaine. Such effects may contribute to the exaggerated neuropathology reported for HIV(+) cocaine-abusing individuals.
Exosomes are small membrane-bound vesicles that carry biological macromolecules from the site of production to target sites either in the microenvironment or at distant sites away from the origin. Exosomal content of cells varies with the cell type that produces them as well as environmental factors that alter the normal state of the cell such as viral infection. Human DNA and RNA viruses alter the composition of host proteins as well as incorporate their own viral proteins and other cargo into the secreted exosomes. While numerous viruses can infect various cell types of the CNS and elicit damaging neuropathologies, few have been studied for their exosomal composition, content, and function on recipient cells. Therefore, there is a pressing need to understand how DNA and RNA viral infections in CNS control exosomal release. Some of the more recent studies including HIV-1, HTLV-1, and EBV-infected B cells indicate that exosomes from these infections contain viral miRNAs, viral transactivators, and a host of cytokines that can control the course of infection. Finally, because exosomes can serve as vehicles for the cellular delivery of proteins and RNA and given that the blood-brain barrier is a formidable challenge in delivering therapeutics to the brain, exosomes may be able to serve as ideal vehicles to deliver protein or RNA-based therapeutics to the brain.
Rift Valley fever virus (RVFV) infection is often associated with pronounced liver damage. Previously, our studies revealed altered host phospho-signaling responses (NF?B, MAPK and DNA damage responses) in RVFV infected epithelial cells that correlated with a cellular stress response. Here, we report that RVFV infection of liver cells leads to an increase in reactive oxygen species (ROS). Our data suggests the presence of the viral protein NSs in the mitochondria of infected cells, hence contributing to early increase in ROS. Increased ROS levels correlated with activation of NF?B (p65) and p53 responses, which in conjunction with infection, was also reflected as macromolecular rearrangements observed using size fractionation of protein lysates. Additionally, we documented an increase in cytokine expression and pro-apoptotic gene expression with infection, which was reversed with antioxidant treatment. Collectively, we identified ROS and oxidative stress as critical contributors to apoptosis of liver cells during RVFV infection.
Exosomes have recently been classified as the newest family members of 'bioactive vesicles' that function to promote intercellular communication. Long ignored and thought to be only a mechanism by which cellular waste is removed, exosomes have garnered a huge amount of interest in recent years as their critical functions in maintaining homeostasis through intercellular communication and also in different types of diseases have been demonstrated. Many groundbreaking studies of exosome functions have been performed in the cancer field and the infectious disease areas of study, revealing the importance and also the fascinating complexity of exosomal packaging, targeting, and functions. Selective packaging of exosomes in response to the type of infection, exosomal modulation of the immune response and host signaling pathways, exosomal regulation of pathogen spread, and effects of exosomes on the degree of pathogenesis have all been well documented. In this review, we provide a synthesis of the current understanding of the role of exosomes during infections caused by human pathogens and discuss the implications of these findings for a better understanding of pathogenic mechanisms and future therapeutic and diagnostic applications.
Most inhibitors of Cyclin-dependent kinase 2 (CDK2) target its ATP-binding pocket. It is difficult, however, to use this pocket to design very specific inhibitors because this catalytic pocket is highly conserved in the protein family of CDKs. Here we report some short peptides targeting a noncatalytic pocket near the interface of the CDK2/Cyclin complex. Docking and molecular dynamics simulations were used to select the peptides, and detailed dynamical network analysis revealed that these peptides weaken the complex formation via allosteric interactions. Our experiments showed that upon binding to the noncatalytic pocket, these peptides break the CDK2/Cyclin complex partially and diminish its kinase activity in vitro. The binding affinity of these peptides measured by Surface Plasmon Resonance can reach as low as 0.5 µM.
HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples.
Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-?B cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-?B complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKK? component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-?B cascade in infected cells, had indicated that the IKK? component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKK? in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKK? function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKK? function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKK? for VEEV replication, we over-expressed IKK? in cells and observed an increase in viral titers. In contrast, studies carried out using IKK?(-/-) cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKK? may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKK? protein may be critically involved in VEEV replication.
The implementation of new antiretroviral therapies targeting transcription of early viral proteins in postintegrated HIV-1 can aid in overcoming current therapy limitations. Using high-throughput screening assays, we have previously described a novel Tat-dependent HIV-1 transcriptional inhibitor named 6-bromoindirubin-3-oxime (6BIO). The screening of 6BIO derivatives yielded unique compounds that show potent inhibition of HIV-1 transcription. We have identified a second-generation derivative called 18BIOder as an inhibitor of HIV-1 Tat-dependent transcription in TZM-bl cells and a potent inhibitor of GSK-3? kinase in vitro. Structurally, 18BIOder is half the molecular weight and structure of its parental compound, 6BIO. More importantly, we also have found a different GSK-3? complex present only in HIV-1-infected cells. 18BIOder preferentially inhibits this novel kinase complex from infected cells at nanomolar concentrations. Finally, we observed that neuronal cultures treated with Tat protein are protected from Tat-mediated cytotoxicity when treated with 18BIOder. Overall, our data suggest that HIV-1 Tat-dependent transcription is sensitive to small-molecule inhibition of GSK-3?.
Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.
Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.
The encoding of microRNAs in retroviral genomes has remained a controversial hypothesis despite significant supporting evidence in recent years. A recent publication demonstrating the production of functional miRNAs from the retrovirus bovine leukemia virus adds further credence to the fact that retroviruses do indeed encode their own miRNAs. Here we comment on the importance of this paper to the field, as well as examine the other known examples of miRNAs encoded by RNA viruses.
Finding insights into how viruses hijack metabolic processes and biomarkers for viral diseases often require hypotheses about target compounds and/or labelling techniques. Here we present a method based on laser ablation electrospray ionization mass spectrometry to rapidly identify potential protein and metabolite biomarkers of oncovirus infection in B lymphocytes.
The Wnt/?-catenin pathway is involved in diverse cell functions governing development and disease. ?-Catenin, a central mediator of this pathway, binds to members of the TCF/LEF family of transcription factors to modulate hundreds of genes. Active Wnt/?-catenin/TCF-4 signaling plays a significant role in repression of HIV-1 replication in multiple cell targets, including astrocytes. To determine the mechanism by which active ?-catenin/TCF-4 leads to inhibition of HIV replication, we knocked down ?-catenin or TCF/LEF members in primary astrocytes and astrocytomas transiently transfected with an HIV long terminal repeat (LTR)-luciferase reporter that contained an integrated copy of the HIV LTR-luciferase construct. Knockdown of either ?-catenin or TCF-4 induced LTR activity by 2- to 3-fold under both the episomal and integrated conditions. This knockdown also increased presence of serine 2-phosphorylated RNA polymerase II (Pol II) on the HIV LTR as well as enhanced its processivity. Knockdown of ?-catenin/TCF-4 also impacted tethering of other transcription factors on the HIV promoter. Specifically, knockdown of TCF-4 enhanced binding of C/EBP?, C/EBP?, and NF-?B to the HIV LTR, while ?-catenin knockdown increased binding of C/EBP? and C/EBP? but had no effect on NF-?B. Approximately 150 genes in astrocytes were impacted by ?-catenin knockdown, including genes involved in inflammation/immunity, uptake/transport, vesicular transport/exocytosis, apoptosis/cellular stress, and cytoskeleton/trafficking. These findings indicate that modulation of the ?-catenin/TCF-4 axis impacts the basal level of HIV transcription in astrocytes, which may drive low level/persistent HIV in astrocytes that can contribute to ongoing neuroinflammation, and this axis also has profound effects on astrocyte biology.
The existence of long-lasting cellular reservoirs of HIV-1 is one of the major hurdles in developing effective anti-retroviral therapies. These latently infected cells and tissues efficiently evade immune responses and remain dormant until activated, upon which they can generate a productive HIV-1 infection. This classic scenario of viral latency becomes even more difficult to study and model due to the extreme complexity of translating in vivo virus-cell interactions into a controlled in vitro system. The recent developments and constant improvements upon hematopoietic engraftment of human cells and tissues onto recipient immunocompromised murine scaffolds have made it possible to model complex human innate and adaptive immune responses in a small animal model. Specifically, HIV-1 infection has been successfully modeled in these humanized mice to mimic transmission, pathogenesis, host immune responses, and treatment. Here, we review the complexities surrounding modeling HIV-1 latency in vitro and in vivo and highlight the most recent humanized mouse models that support retroviral infection.
HIV-1 is a retrovirus that has infected millions in recent decades. The level of life cycle complexity and host control exerted by this small virus with only nine proteins is astonishing. An interesting direction that has emerged in recent years is the role of small non-coding RNAs in viral gene expression.
The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the hosts fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.
To establish whether activation of adenosine type-3 receptors (A3Rs) and inhibition of interleukin-1?-induced inflammation is beneficial in combination with antibiotic therapy to increase survival of mice challenged with anthrax spores.
The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified ?-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.
The human immunodeficiency virus type 1 (HIV-1) long terminal repeat is present on both ends of the integrated viral genome and contains regulatory elements needed for transcriptional initiation and elongation. Post-integration, a highly ordered chromatin structure consisting of at least five nucleosomes, is found at the 5 long terminal repeat, the location and modification state of which control the state of active viral replication as well as silencing of the latent HIV-1 provirus. In this context, the chromatin remodeling field rapidly emerges as having a critical role in the control of viral gene expression. In the current study, we focused on unique Baf subunits that are common to the most highly recognized of chromatin remodeling proteins, the SWI/SNF (switching-defective-sucrose non-fermenting) complexes. We find that at least two Baf proteins, Baf53 and Baf170, are highly regulated in HIV-1-infected cells. Previously, studies have shown that the depletion of Baf53 in uninfected cells leads to the expansion of chromosomal territories and the decompaction of the chromatin. Baf53, in the presence of HIV-1 infection, co-elutes off of a chromatographic column as a different-sized complex when compared to uninfected cells and appears to be predominantly phosphorylated. The innate function of Baf53-containing complexes appears to be transcriptionally suppressive, in that knocking down Baf53 increases viral gene expression from cells both transiently and chronically infected with HIV-1. Additionally, cdk9/cyclin T in the presence of Tat is able to phosphorylate Baf53 in vitro, implying that this posttranslationally modified form relieves the suppressive effect and allows for viral transcription to proceed.
Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNF? following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells.
the search for effective inhibitors to multiple infectious agents including influenza, smallpox and hemorrhagic fever viruses is an area of active research as many of these agents pose dramatic health and economic challenges to the human population. Many of these infectious agents are not only endemic threats in different parts of the globe, but are also considered to have the potential of being used as bioterrorism agents.
Extracellular iron has been implicated in the pathogenesis of post-injury organ failure. However, the source(s) and biochemical species of this iron have not been identified. Based upon evidence that distant organ injury results from an increase in intestinal permeability, we looked for ferrous iron in mesenteric lymph in anesthetized rats undergoing hemorrhage and fluid resuscitation (H/R). Ferrous iron increased in lymph from 4.7 nmol/mg of protein prior to hemorrhage to 86.6 nmol/mg during resuscitation. Utilizing immuno-spin trapping in protein fractions that were rich in iron, we tentatively indentified protein carrier(s) of ferrous iron by MALDI-TOF MS. One of the identified proteins was the metalloproteinase (MMP) inhibitor, TIMP-2. Antibody to TIMP-2 immunoprecipitated 74% of the ferrozine detectable iron in its protein fraction. TIMP-2 binds iron in vitro at pH 6.3, which is typical of conditions in the mesentery during hemorrhage, but it retains the ability to inhibit the metalloproteases MMP-2 and MMP-9. In summary, there is a large increase in extracellular ferrous iron in the gut in H/R demonstrating dysregulation of iron homeostasis. We have identified, for the first time, the binding of extracellular iron to TIMP-2.
The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC(50) of 40nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC(50) of 4nM in primary macrophages and 0.5nM in astrocytes infected with HIV-1. 6BIOder displayed an IC(50) value of 0.03nM through in vitro GSK-3? kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity.
HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670-7675, 2006; J Med Chem 50:3716-3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-?B-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs.
Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells.
MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the hosts miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.
Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF-?B phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication.
Approximately half of hereditary breast cancers have mutations in either BRCA1 or BRCA2. BRCA1 is a multifaceted tumor suppressor protein that has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. This multifunctional nature of BRCA1 is achieved by exerting its many effects through modulation of transcription. Many cellular events are dictated by covalent modification of proteins, an important mechanism in regulating protein and genome function; of which protein methylation is an important posttranslational modification with activating or repressive effects.
Cholesterol plays an important role in the HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits the activity of the major cellular cholesterol transporter ATP binding cassette transporter A1 (ABCA1), suggesting that the virus may use this mechanism to get access to cellular cholesterol. In this study, we investigated the effect on HIV infection of a synthetic liver X receptor (LXR) ligand, N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (TO-901317), which is a potent stimulator of ABCA1 expression. We demonstrate that TO-901317 restores cholesterol efflux from HIV-infected T lymphocytes and macrophages. TO-901317 potently suppressed HIV-1 replication in both cell types and inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in RAG-hu mice infected in vivo. This anti-HIV activity was dependent on ABCA1, because the effect of the drug was significantly reduced in ABCA1-defective T cells from a patient with Tangier disease, and RNA interference-mediated inhibition of ABCA1 expression eliminated the effect of TO-901317 on HIV-1 replication. TO-901317-mediated inhibition of HIV replication was due to reduced virus production and reduced infectivity of produced virions. The infectivity defect was in part due to reduced fusion activity of the virions, which was directly linked to reduced viral cholesterol. These results describe a novel approach to inhibiting HIV infection by stimulating ABCA1 expression.
Current therapy for human immunodeficiency virus (HIV-1) infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI) r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202) in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs.
Physiological regulation of cellular iron involves iron export by the membrane protein, ferroportin, the expression of which is induced by iron and negatively modulated by hepcidin. We previously showed that iron chelation is associated with decreased HIV-1 transcription. We hypothesized that increased iron export by ferroportin might be associated with decreased HIV-1 transcription, and degradation of ferroportin by hepcidin might in turn induce HIV-1 transcription and replication. Here, we analyzed the effect of ferroportin and hepcidin on HIV-1 transcription.
RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the hosts response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.
The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patients cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum.
Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF-kappaB. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3 end of the genome. Because of this, a variety of chromatin remodelers can act to modify nucleosomes, allowing for efficient transcription. While a number of covalent modifications are known to occur on histone tails in HTLV-1 infection (i.e., histone acetyltransferases [HATs], histone deacetylases [HDACs], and histone methyltransferases [HMTs]), evidence points to the use of chromatin remodelers that use energy from ATP hydrolysis to remodel nucleosomes. Here we confirm that BRG1, which is the core subunit of eight chromatin-remodeling complexes, is essential not only for Tax transactivation but also for viral replication. This is especially evident when wild-type infectious clones of HTLV-1 are used. BRG1 associates with Tax at the HTLV-1 long terminal repeat (LTR), and coexpression of BRG1 and Tax results in increased rates of transcription. The interaction of BRG1 with Tax additionally recruits the basal transcriptional machinery and removes some of the core histones from the nucleosome at the start site (Nuc 1). When using the BRG1-deficient cell lines SW13, C33A, and TSUPR1, we observed little viral transcription and no viral replication. Importantly, while these three cell lines do not express detectable levels of BRG1, much of the SWI/SNF complex remains assembled in the cells. Knockdown of BRG1 and associated SWI/SNF subunits suggests that the BRG1-utilizing SWI/SNF complex PBAF is responsible for HTLV-1 nucleosome remodeling. Finally, HTLV-1 infection of cell lines with a knockdown in BRG1 or the PBAF complex results in a significant reduction in viral production. Overall, we concluded that BRG1 is required for Tax transactivation and HTLV-1 viral production and that the PBAF complex appears to be responsible for nucleosome remodeling.
The SWI/SNF complex remodels nucleosomes, allowing RNA Polymerase II access to the HIV-1 proviral DNA. It has not been determined which SWI/SNF complex (BAF or PBAF) remodels nucleosomes at the transcription start site. These complexes differ in only three subunits and determining which subunit(s) is required could explain the regulation of Tat activated transcription. We show that PBAF is required for chromatin remodeling at the nuc-1 start site and transcriptional elongation. We find that Baf200 is required to ensure activation at the LTR level and for viral production. Interestingly, the BAF complex was observed on the LTR whereas PBAF was present on both LTR and Env regions. We found that Tat activated transcription facilitates removal of histones H2A and H2B at the LTR, and that the FACT complex may be responsible for their removal. Finally, the BAF complex may play an important role in regulating splicing of the HIV-1 genome.
HIV-1 is a small retrovirus that wreaks havoc on the human immune system. It is a puzzle to the scientific community how a virus that encodes only nine proteins can take complete control of its host and redirect the cell to complete replication or maintain latency when necessary. One way to explain the control elicited by HIV-1 is through numerous protein partners that exist between viral and host proteins, allowing HIV-1 to be intimately involved in virtually every aspect of cellular biology. In addition, we postulate that the complexity exerted by HIV-1 can not merely be explained by the large number of protein-protein interactions documented in the literature but, rather, cell-type-specific interactions and post-translational modifications of viral proteins must be taken into account. We use HIV-1 Tat and its influence on viral transcription as an example of cell-type-specific complexity. The influence of post-translational modifications (acetylation and methylation), as well as subcellular localization on Tat binding partners, is also discussed.
The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse transcriptase, viral protease or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we discuss how targeting the host can be accomplished either by using small molecules to alter the function of the hosts proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be used to better design therapeutics against HIV-1.
HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-kappaB, and others. The cells of most of the bodys organs are exposed to approximately 3-6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O(2), and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O(2) compared to 21% O(2). HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O(2) compared to 21% O(2). This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O(2), the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-kappaB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O(2) and also expression of CDK9/cyclin T1-dependent IkappaB inhibitor alpha was repressed. Our results suggest that lower HIV-1 transcription at 3% O(2) compared to 21% O(2) may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O(2) and that additional host cell factors such as CDK2 and NF-kappaB might be major regulators of HIV-1 transcription at low O(2) concentrations.
Most viral treatments target the virus itself, providing very specific effects and limiting side-effects on uninfected cells. However, this strategy of drug design often results in resistant viruses, especially among RNA viruses. Therefore, the focus has turned to drugs that target cellular proteins that are essential for viral replication, but not for cellular viability. Pharmacological CDK inhibitors are a prime example of this type of approach. Reviewed within are the various functions of CDKs, their role in the life cycle of selected Retroviruses and Herpesviruses, and the pharmacological CDK inhibitors that have been focused on in terms of viral inhibition.
As part of a continued search for more efficient anti-HIV-1 drugs, we are focusing on the possibility that small molecules could efficiently inhibit HIV-1 replication through the restoration of p53 and p21WAF1 functions, which are inactivated by HIV-1 infection. Here we describe the molecular mechanism of 9-aminoacridine (9AA) mediated HIV-1 inhibition. 9AA treatment resulted in inhibition of HIV LTR transcription in a specific manner that was highly dependent on the presence and location of the amino moiety. Importantly, virus replication was found to be inhibited in HIV-1 infected cell lines by 9AA in a dose-dependent manner without inhibiting cellular proliferation or inducing cell death. 9AA inhibited viral replication in both p53 wildtype and p53 mutant cells, indicating that there is another p53 independent factor that was critical for HIV inhibition. p21WAF1 is an ideal candidate as p21WAF1 levels were increased in both p53 wildtype and p53 mutant cells, and p21WAF1 was found to be phosphorylated at S146, an event previously shown to increase its stability. Furthermore, we observed p21WAF1 in complex with cyclin T1 and cdk9 in vitro, suggesting a direct role of p21WAF1 in HIV transcription inhibition. Finally, 9AA treatment resulted in loss of cdk9 from the viral promoter, providing one possible mechanism of transcriptional inhibition. Thus, 9AA treatment was highly efficient at reactivating the p53 - p21WAF1 pathway and consequently inhibiting HIV replication and transcription.
Chromatin remodeling is an essential event for HIV-1 transcription. Over the last two decades this field of research has come to the forefront, as silencing of the HIV-1 provirus through chromatin modifications has been linked to latency. Here, we focus on chromatin remodeling, especially in relation to the transactivator Tat, and review the most important and newly emerging studies that investigate remodeling mechanisms. We begin by discussing covalent modifications that can alter chromatin structure including acetylation, deacetylation, and methylation, as well as topics addressing the interplay between chromatin remodeling and splicing. Next, we focus on complexes that use the energy of ATP to remove or secure nucleosomes and can additionally act to control HIV-1 transcription. Finally, we cover recent literature on viral microRNAs which have been shown to alter chromatin structure by inducing methylation or even by remodeling nucleosomes.
The development of novel techniques and systems to study human infectious diseases in both an in vitro and in vivo settings is always in high demand. Ideally, small animal models are the most efficient method of studying human afflictions. This is especially evident in the study of the human retroviruses, HIV-1 and HTLV-1, in that current simian animal models, though robust, are often expensive and difficult to maintain. Over the past two decades, the construction of humanized animal models through the transplantation and engraftment of human tissues or progenitor cells into immunocompromised mouse strains has allowed for the development of a reconstituted human tissue scaffold in a small animal system. The utilization of small animal models for retroviral studies required expansion of the early CB-17 scid/scid mouse resulting in animals demonstrating improved engraftment efficiency and infectivity. The implantation of uneducated human immune cells and associated tissue provided the basis for the SCID-hu Thy/Liv and hu-PBL-SCID models. Engraftment efficiency of these tissues was further improved through the integration of the non-obese diabetic (NOD) mutation leading to the creation of NODSCID, NOD/Shi-scid IL2rgamma-/-, and NOD/SCID beta2-microglobulinnull animals. Further efforts at minimizing the response of the innate murine immune system produced the Rag2-/-gammac-/- model which marked an important advancement in the use of human CD34+ hematopoietic stem cells. Together, these animal models have revolutionized the investigation of retroviral infections in vivo.
RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression.
The cyclin-dependent kinase 2 (cdk2) is a serine/threonine protein kinase that plays a key role in the cell cycle control system of all eukaryotic organisms. It has been a much studied drug target for potential anticancer therapy. Most cdk2 inhibitors in clinical development target almost exclusively the catalytic ATP-binding pocket of cdk2. However, several five amino-acid peptide inhibitors that are directed towards a noncatalytic binding pocket of cdk2 are reported here. Upon binding to this new pocket located at the cdk2 and cyclin interface, these peptide inhibitors are found to disrupt the cdk2/cyclin E complex partially and diminish its kinase activity in vitro.
Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.
Human T-lymphotropic virus 1 (HTLV-1) was the first human retrovirus to be discovered and is the causative agent of adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The importance of microRNA (miRNA) in the replicative cycle of several other viruses, as well as in the progression of associated pathologies, has been well established in the past decade. Moreover, involvement of miRNA alteration in the HTLV-1 life cycle, and in the progression of its related oncogenic and neurodegenerative diseases, has recently come to light. Several HTLV-1 derived proteins alter transcription factor functionalities, interact with chromatin remodelers, or manipulate components of the RNA interference (RNAi) machinery, thereby establishing various routes by which miRNA expression can be up- or down-regulated in the host cell. Furthermore, the mechanism of action through which dysregulation of host miRNAs affects HTLV-1 infected cells can vary substantially and include mRNA silencing via the RNA-induced silencing complex (RISC), transcriptional gene silencing, inhibition of RNAi components, and chromatin remodeling. These miRNA-induced changes can lead to increased cell survival, invasiveness, proliferation, and differentiation, as well as allow for viral latency. While many recent studies have successfully implicated miRNAs in the life cycle and pathogenesis of HTLV-1 infections, there are still significant outstanding questions to be addressed. Here we will review recent discoveries elucidating HTLV-1 mediated manipulation of host cell miRNA profiles and examine the impact on pathogenesis, as well as explore future lines of inquiry that could increase understanding in this field of study.
Potent anti-retroviral therapy has transformed HIV-1 infection into a chronic manageable disease; however, drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study, we utilized a combination of structure-based analysis of cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket that showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell-based biological assays. Using these methods, we obtained the first-generation mimetic drugs and tested these compounds on HIV-1 long terminal repeat-activated transcription. Using biological assays followed by similar in silico analysis to find second-generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the second-generation mimetic against various viral isolates and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2(-/-)?c(-/-) with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis.
Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2-5% develop ATL. The past 35?years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-?B pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis.
Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. N?-nitro-L-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.
HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity.
Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%-60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyers patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.
Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NF?B cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of I?B? and occurs through the classical NF?B cascade. A unique, low molecular weight complex of the IKK-? subunit can be observed in MP-12-infected cells, which we have labeled IKK-?2. The IKK-?2 complex retains kinase activity and phosphorylates an I?B? substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-?2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-?2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-?2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.
HIV-1 proteins, including the transactivator of transcription (Tat), are believed to be involved in HIV-associated neurocognitive disorders by disrupting Ca²? homeostasis, which leads to progressive dysregulation, damage, or death of neurons in the brain. We have found previously that bath-applied Tat abnormally increased Ca²? influx through overactivated, voltage-sensitive L-type Ca²? channels in pyramidal neurons within the rat medial prefrontal cortex (mPFC). However, it is unknown whether the Tat-induced Ca²? dysregulation was mediated by increased activity and/or the number of the L-channels. This study tested the hypothesis that transient/early exposure to Tat in vivo promoted enduring L-channel dysregulation in the mPFC without neuron loss. Accordingly, rats were administered a single intracerebroventricular injection of recombinant Tat (80 ?g/20 ?l; diluted by cerebrospinal fluids to pathophysiological concentrations) or vehicle. Rats were killed 14 days after injection for immunohistochemical assessments of the mPFC, motor cortex, caudate-putamen, and nucleus accumbens. Stereological estimates for positively stained cells indicated a significant increase in the number of cells expressing the pore-forming Ca(v)1.2-?1c subunit of L-channels in the mPFC compared with other regions in Tat-treated or vehicle-treated rat brains. Optical density measurements showed a Tat-induced increase in glial fibrillary acidic protein expression, indicating astrogliosis in the cortical regions. There was no significant loss of neurons in any brain region investigated. These findings indicate that transient Tat exposure in vivo induced enduring L-channel dysregulation and astrogliosis in the mPFC without neuron loss. Such maladaptations may contribute toward dysregulated Ca²? homeostasis and neuropathology in the PFC in the early stages of HIV infection.
The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs, suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against present day retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack both the enzymatic and catalytic components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active viral infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components of this pathway. A survey of uninfected and HTLV-1 infected cells revealed that Drosha protein is present at lower levels in all HTLV-1 infected cell lines and in infected primary cells, while other components such as DGCR8 were not dramatically altered. We show colocalization of Tax and Drosha in the nucleus in vitro as well as coimmunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus.
Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of "latent cell populations" even after Anti-Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells.
Molecular regulation of HIV transcription is a multifaceted process dictated in part by the abundance of cellular transcription factors that induce or repress HIV promoter activity. ?-Catenin partners with members of the T cell factor (TCF)/LEF transcription factors to regulate gene expression. The interaction between ?-catenin and TCF-4 is linked to inhibition of HIV replication in multiple cell types, including lymphocytes and astrocytes. Here, we evaluated the molecular mechanism by which ?-catenin/TCF-4 repress HIV replication. We identified for the first time multiple TCF-4 binding sites at -336, -143, +66, and +186 relative to the transcription initiation site on the HIV long terminal repeat (LTR). Two of the sites (-143 and +66) were present in approximately 1/3 of 500 HIV-1 isolates examined. Although all four sites could bind to TCF-4, the strongest association occurred at -143. Deletion and/or mutation of -143, in conjunction with ?-catenin or TCF-4 knockdown in cells stably expressing an LTR reporter construct, enhanced basal HIV promoter activity by 5-fold but had no effect on Tat-mediated transactivation of the HIV LTR. We also found that TCF-4, ?-catenin, and the nuclear matrix binding protein SMAR1 tether at the -143-nucleotide (nt) site on the HIV LTR to inhibit HIV promoter activity. Collectively, these data indicate that TCF-4 and ?-catenin at -143 associate with SMAR1, which likely pulls the HIV DNA segment into the nuclear matrix and away from transcriptional machinery, leading to repression of basal HIV LTR transcription. These studies point to novel avenues for regulation of HIV replication by manipulation of ?-catenin signaling within cells.
Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production.
Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-?, IL-6, IL-12, iNOS and TNF-?) within 6 h. GSK-3? is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimers. Hence inhibition of GSK-3? in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3? inhibitors and GSK-3? siRNA experiments indicated that GSK-3? was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC(50) of ?0.5 µM and a CC(50) of >100 µM. BIOder was a more potent inhibitor of GSK-3? than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3? is found in three distinct complexes in VEEV infected cells, whereas GSK-3? is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3? inhibitors for modulating VEEV infection.
To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacterias ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ?inhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.
HIV-infected subjects are at high risk of developing atherosclerosis, in part due to virus-induced impairment of HDL metabolism. Here, using as a model of HIV infection the NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/SzJ (NSG) mice humanized by human stem cell transplantation, we demonstrate that LXR agonist TO901317 potently reduces viral replication and prevents HIV-induced reduction of plasma HDL. These results establish that humanized mice can be used to investigate the mechanisms of HIV-induced impairment of HDL formation, a major feature of dyslipidemia associated with HIV-1 infection, and show potential benefits of developing LXR agonists for treatment of HIV-associated cardio-vascular disease.
Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.