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Fusion Lifecycle 2014 Activation |TOP|


Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity.




Fusion Lifecycle 2014 Activation



Importance: After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.


Gene activation by dCas9, also referred to as CRISPRa, was initially published in 2013 (Bikard et al., 2013, Perez-Pinera et al., 2013). In the years that followed, innovative methods greatly improved CRISPRa, expanding its practicality and popularity in research (Tanenbaum et al., 2014, Konermann et al., 2015, Chavez et al., 2015).


CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability, to bind to genes without editing the genome (Qi et al., 2013). To target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all shown significant improvements upon the initial dCas9-VP64 method, so there are multiple options to choose from when looking to activate genes across diverse cell lines.


SAM uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 fusion protein engineered with aptamers that bind to MS2 proteins. These MS2 proteins then recruit additional activation domains (HS1 and p65).


Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator, but lower levels compared to the SAM system. Its activation levels are similar to that of SunTag. An advantage to this method compared to other notable CRISPR activators is that it requires a fusion protein, rather than relying on a two-component system dependent on gRNA design (SAM) or peptide design (SunTag). This streamlines its delivery, making it a common choice for CRISPR activation.


Citation: Dominguez PM and Shaknovich R (2014) Epigenetic function of activation-induced cytidine deaminase and its link to lymphomagenesis. Front. Immunol. 5:642. doi: 10.3389/fimmu.2014.00642


PTS also can be regulated by photoactivation, either by intein fusion to a photodimerization domain[86] or by the addition of protecting groups that are photo-cleavable[87, 88], as reviewed in[7]. More recently, Mootz and coworkers have designed a split Ssp DnaB intein than can induce C-terminal cleavage on irradiation. They used this system to liberate staphylocoagulase from the IC segment, which in turn activated native prothrombin, both in vitro and in plasma[89]. Protein splicing side reactions may also be enhanced by changes in pH[90].


Intein biosensors for protein-protein interactions utilize PTS facilitated by split intein fragments that have low binding affinity for each other. Design of these biosensors involves creation of two fusion proteins, each containing one protein binding partner, a split intein fragment and a fragment of a reporter protein (Figure 6B). Interaction of the binding partners facilitates split intein reconstitution and splicing-induced complementation and activation of a reporter protein. Umezawa and coworkers applied this sensor design to demonstrate protein-protein interactions in various in vivo systems ranging from E. coli to transgenic animals. In their original work, an E. coli-based biosensor was developed to monitor binding between calmodulin and its target peptide M13, using GFP reconstitution as the reporter, mediated by the artificially split Sce VMAI intein[99]. Next, an insulin-induced interaction between phosphorylated insulin receptor substrate 1 and its target (the N-terminal SH2 domain of PI 3-kinase) was observed in mammalian cells by luciferase reconstitution by the naturally split Ssp DnaE intein[100]. Then, they demonstrated a bioluminescence imaging method to noninvasively and quantitatively image protein-protein interactions in mice by intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id[101]. To increase the sensitivity of detection, protein splicing was employed to produce a functional transcription factor that modulates a reporter gene, firefly luciferase[102, 103]. In this work, the epidermal growth factor (EGF)-induced interactions of an oncogenic product Ras and its target Raf-1 was monitored by bioluminescence signals in mammalian cells. Notably, this interaction was not detected by traditional two-hybrid systems.


A protein localization-dependent sensor also was developed for corticosterone detection in animals[109]. Again, the biosensor has two components. The first is a cytosol-localized fusion of glucocorticoid receptor with C-terminal fragments of the Ssp DnaE intein and split luciferase. The second is a nucleus-localized fusion of the N-terminal fragments of the intein and luciferase. Upon corticosterone binding, the glucocorticoid receptor is translocated into the nucleus, facilitating intein fragment complementation and splicing and therefore activation of luciferase.


Early studies characterizing Parkin- or PINK1-null Drosophila mutants found evidence of swollen mitochondria in numerous tissue [50,51,52, 118, 119], suggesting that PINK1 and Parkin may either drive fission or inhibit fusion. These pathological features could be ameliorated by increasing expression of Drp1 or reducing Opa1 or Mitofusin [120,121,122,123], suggesting a genetic interaction between PINK1/Parkin and canonical fission/fusion regulatory pathways. Both PINK1 and Parkin promote degradation of critical mitochondrial fusion proteins Mitofusin 1 and 2 [72, 124,125,126,127,128,129,130,131], while PINK1 is sufficient to promote mitochondrial fission by recruiting Drp1 to mitochondria [132, 133]. Thus, Parkin/PINK1 activation seems to drive mitochondrial dynamics towards fission by activating pro-fission and inactivating pro-fusion pathways (Fig. 1b). PINK1/Parkin-induced mitochondrial fission potentially contributes to MQC through two parallel mechanisms. First, it could act to segregate areas of focal damage. For example, a recent study in HeLa cells used mutant, aggregation-prone ornithine transcarbamylase (OTC) to induce misfolded protein foci in mitochondria [134]. These foci led to local accumulation of PINK1/Parkin and subsequent OTC clearance by mitochondrial fission [134]. Ablating mitochondrial fission through Drp1-KO did not affect the rate of OTC clearance; instead, it lead to generalized recruitment of PINK1/Parkin and substantial upregulation of mitophagy [134]. Thus, in the case of focal damage, mitochondrial fission appears to be an initial defense which enables selective removal of dysfunctional components, thereby preventing mitophagy from destroying healthy mitochondria. However, this process relies on focal concentrations of mitochondrial damage. It is unclear if and how mitochondrial fission could contribute to the management of more diffuse damage or to baseline MQC in the absence of an insult.


Early studies have long implicated selective vulnerability of SNpc DA neurons and mitochondrial dysfunction as core features of PD. While we now understand that PD processes are far more distributed across the CNS and may be driven primarily by prion-like mechanisms spreading α-synuclein aggregates, these non-cell autonomous mechanisms likely act in concert with cell- and region-specific factors that lead to selective vulnerability to neurodegeneration. Though these cell-intrinsic factors are likely complex and varied across the different vulnerable subpopulations, in SNpc DA neurons findings over the last few decades point to the unique mitochondrial challenges and stresses due to complex cytoarchitecture as a potential major cause. Probing the function of PINK1/Parkin has led to critical insights into their role in maintaining mitochondrial integrity and proteostasis in the face of the stressors faced by mitochondria. These protective mechanisms comprise multiple tiers of MQC, such as facilitating mitophagy, regulating fission/fusion dynamics, triggering removal of damaged mitochondrial components through MDV generation, promoting mitochondrial biogenesis by increasing PGC-1α, and regulating the local translation of mitochondrial genes (Fig. 1), though many of these proposed mechanisms require convincing validation in the mammalian CNS. Newer areas of research have begun to establish mechanisms by which α-synuclein aggregation causes inactivation of MQC (Fig. 2), which have clear implications for sPD, as well as how MQC defects in neurons and non-neuronal cells may contribute to neuroimmune mechanisms of neurodegeneration.


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