scholarly journals Molecular Mechanisms Controlling HIV Transcription and Latency – Implications for Therapeutic Viral Reactivation

Author(s):  
Michael D. Rölling ◽  
Mateusz Stoszko ◽  
Tokameh Mahmoudi
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor

AbstractHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.


2021 ◽  
Vol 22 (12) ◽  
pp. 6279
Author(s):  
Giorgia Napoletani ◽  
Virginia Protto ◽  
Maria Elena Marcocci ◽  
Lucia Nencioni ◽  
Anna Teresa Palamara ◽  
...  

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer’s disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.


2020 ◽  
Vol 117 (34) ◽  
pp. 20860-20867 ◽  
Author(s):  
Benjamin A. Krishna ◽  
Amanda B. Wass ◽  
Christine M. O’Connor

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G protein-coupled receptorUS28is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1–independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.


2008 ◽  
Vol 82 (24) ◽  
pp. 12291-12303 ◽  
Author(s):  
Richard Pearson ◽  
Young Kyeung Kim ◽  
Joseph Hokello ◽  
Kara Lassen ◽  
Julia Friedman ◽  
...  

ABSTRACT The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP+ and d2EGFP− cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP+ cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes IκBα and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-α associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.


Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.


Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.


Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.


2020 ◽  
Vol 48 (3) ◽  
pp. 1019-1034 ◽  
Author(s):  
Rachel M. Woodhouse ◽  
Alyson Ashe

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.


2020 ◽  
Vol 48 (2) ◽  
pp. 429-439 ◽  
Author(s):  
Jorge Gago ◽  
Danilo M. Daloso ◽  
Marc Carriquí ◽  
Miquel Nadal ◽  
Melanie Morales ◽  
...  

Besides stomata, the photosynthetic CO2 pathway also involves the transport of CO2 from the sub-stomatal air spaces inside to the carboxylation sites in the chloroplast stroma, where Rubisco is located. This pathway is far to be a simple and direct way, formed by series of consecutive barriers that the CO2 should cross to be finally assimilated in photosynthesis, known as the mesophyll conductance (gm). Therefore, the gm reflects the pathway through different air, water and biophysical barriers within the leaf tissues and cell structures. Currently, it is known that gm can impose the same level of limitation (or even higher depending of the conditions) to photosynthesis than the wider known stomata or biochemistry. In this mini-review, we are focused on each of the gm determinants to summarize the current knowledge on the mechanisms driving gm from anatomical to metabolic and biochemical perspectives. Special attention deserve the latest studies demonstrating the importance of the molecular mechanisms driving anatomical traits as cell wall and the chloroplast surface exposed to the mesophyll airspaces (Sc/S) that significantly constrain gm. However, even considering these recent discoveries, still is poorly understood the mechanisms about signaling pathways linking the environment a/biotic stressors with gm responses. Thus, considering the main role of gm as a major driver of the CO2 availability at the carboxylation sites, future studies into these aspects will help us to understand photosynthesis responses in a global change framework.


2020 ◽  
Vol 134 (17) ◽  
pp. 2243-2262
Author(s):  
Danlin Liu ◽  
Gavin Richardson ◽  
Fehmi M. Benli ◽  
Catherine Park ◽  
João V. de Souza ◽  
...  

Abstract In the elderly population, pathological inflammation has been associated with ageing-associated diseases. The term ‘inflammageing’, which was used for the first time by Franceschi and co-workers in 2000, is associated with the chronic, low-grade, subclinical inflammatory processes coupled to biological ageing. The source of these inflammatory processes is debated. The senescence-associated secretory phenotype (SASP) has been proposed as the main origin of inflammageing. The SASP is characterised by the release of inflammatory cytokines, elevated activation of the NLRP3 inflammasome, altered regulation of acetylcholine (ACh) nicotinic receptors, and abnormal NAD+ metabolism. Therefore, SASP may be ‘druggable’ by small molecule therapeutics targeting those emerging molecular targets. It has been shown that inflammageing is a hallmark of various cardiovascular diseases, including atherosclerosis, hypertension, and adverse cardiac remodelling. Therefore, the pathomechanism involving SASP activation via the NLRP3 inflammasome; modulation of NLRP3 via α7 nicotinic ACh receptors; and modulation by senolytics targeting other proteins have gained a lot of interest within cardiovascular research and drug development communities. In this review, which offers a unique view from both clinical and preclinical target-based drug discovery perspectives, we have focused on cardiovascular inflammageing and its molecular mechanisms. We have outlined the mechanistic links between inflammageing, SASP, interleukin (IL)-1β, NLRP3 inflammasome, nicotinic ACh receptors, and molecular targets of senolytic drugs in the context of cardiovascular diseases. We have addressed the ‘druggability’ of NLRP3 and nicotinic α7 receptors by small molecules, as these proteins represent novel and exciting targets for therapeutic interventions targeting inflammageing in the cardiovascular system and beyond.


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