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Live cell imaging of single genomic loci with quantum dot-labeled TALEs
Daiwen Pang Wuhan University and Prof. Qinxue Hu Wuhan Institute of Virology. This work is licensed under a Creative Commons Attribution 4. Retrovirology By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
Article metrics. Advanced search. Skip to main content. Subjects DNA Molecular imaging. Abstract Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Introduction Single genomic loci in human chromosomes are often related to specific cellular functions, human genetic diseases and pathogen infections. Full size image. Figure 6: Imaging of single gene loci in live U1 cells. Discussion In summary, we have reported a novel approach to allow the visualization of single genomic loci in live cells.
Cell culture and transfection U1 provided by Prof.
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Data availability The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information , or from its author upon reasonable request. Additional information How to cite this article: Ma, Y. References 1. Google Scholar 2. Google Scholar 4. Google Scholar 5. Despite lingering safety concerns [ 1 ] and potential restrictions imposed by the host immune response, including the innate immune pattern recognition receptors PRR , replication-defective and conditionally replicating human and nonhuman adenovirus AdV vectors continue to be a favorite vehicle for short-term e.
This is due in part to several desirable features of AdV including their broad tissue tropism, their ample capacity for foreign gene insertion, and their LEGO-like structural adaptability Fig 1A to add, delete, and swap proteins and motifs from other viruses or host molecules. A An illustration of the cross-section of a prototype 90 nm AdV capsid showing the location of the principal capsid proteins hexon, penton, protein VI, protein IX, protease, and the fibre—the knob is the globular head of the fibre involved in trafficking.
B An illustration showing the quintessential steps of AdV trafficking in epithelial cells. Via the knob region of the fiber, the capsid engages the cellular receptor. Postinternalization, the capsid continues to dissociate and releases protein VI, which allows the capsid access to the cytosol and interaction with dynein, then dynein-dependent transport along microtubule to the nuclear pore complex.
Overall, the particle is stable to the environment; however, it is able to respond to cellular cues to undergo conformational changes during cell entry. While much is known about the molecular genetics and replication of AdVs, many investigators are continuing to decipher the captivating intracellular events of the first thirty minutes in the virus life cycle. Similar viral and cellular proteins are used, and although the function of the cellular protein varies among cell types, these cell protein—virus associations promote similar outcomes. We also highlight some outstanding questions and hurdles needed to improve vector-mediated gene and vaccine delivery and treatments for AdV disease.
The take home message is that one may be able to take advantage of a better understanding of these cell entry variations to control AdV pathogenesis and vector tropism for gene therapy. Many human and some nonhuman AdVs, including CAV-2, use the coxsackievirus and adenovirus receptor CAR [ 2 — 4 ] for high affinity attachment to host cells via the capsid fibre protein. On polarized epithelial cells, the predominant CAR isoform is targeted to the basolateral surface and in tight junctions.
A minor exon 8-containing CAR isoform may be targeted to the apical surface [ 5 ] of some epithelial cells and allow easier access of CAR-tropic virus attachment. This engagement occurs through association of the integrin with a consensus integrin interacting motif RGD in most AdVs located on an extended loop on the penton base [ 7 ]. Integrin ligation triggers signaling events that promote virus entry into early endosomes via clathrin-mediated endocytosis Fig 1B. In epithelial cells, it seems that CAR facilitates attachment but not cell entry [ 8 ]. However, it is still unclear how significantly the integrin repertoire involved in membrane penetration influences different AdV types.
Moreover, when injected intravenously in mice, some AdVs can interact with specific coagulation factors [ 9 ] that alter tissue tropism by preventing binding of naturally occurring antibodies and then by acting as a bridge to attach to proteoglycans on liver cells [ 10 ]. As discussed below, AdV trafficking into neurons follows a pathway different from that of epithelial cells.
Thus, the routes and modes of AdV cell entry are variable and cell-type dependent. Internalization of AdV particles is a primordial event for infection—but it is only the beginning of the journey to the nuclear pore complex NPC. The ligation of CAR and integrins on the cell surface induces distinct membrane trafficking processes that produce a mechanical force to initiate partial capsid disassembly [ 11 ]. Analyses using atomic force microscopy are consistent with this model and indicate that integrin ligation by the virus is sufficient to loosen the vertex region s of the capsid [ 12 ].
Removal of the vertex region—composed of the penton base, fibre [ 13 ], and likely the peripentonal hexons—allows release of the membrane lytic protein VI from the inner surface of the virus capsid [ 14 ]. This restricts release of protein VI and membrane destruction. Exposure of the inner core of the virus apparently starts in the endosome as monitored by antibody detection of the viral genome at early time points [ 15 ]. Protein VI release is associated with increased endosomal membrane destruction [ 13 ], and a single point mutation L40Q in the amphipathic helical domain of protein VI significantly attenuates membrane insertion, membrane destruction, and cell infection [ 16 ].
Protein VI insertion into a lipid bilayer causes positive membrane curvature [ 17 ], and this may impart stress and global membrane destruction allowing passage of the partially disassembled capsid into the cytosol. If there are structural rearrangements that occur in protein VI after its release from the HAdV capsid, these could be druggable targets. Partially uncoated virions also escape endocytic vesicle concomitant with a drop in pH. Partially uncoated virions can associate with dynein motor proteins that recognize the hexon [ 19 ] or possibly protein VI [ 20 ].
This association is instrumental in transporting the virions along microtubules, and numerous laboratories have seen that, in superinfected cells, AdV capsids can accumulate at the microtubule-organizing center. However, it is unclear whether the microtubule-organizing center is a launching pad for NPC engagement, an artifact of superinfected cells, or a cul-de-sac. The association of the virion with proteins at the NPC likely facilitates further uncoating of the virion, thereby allowing the genome to be translocated into the nucleus, although the precise mechanisms involved are still being investigated.
Afterwards, the AdV genome is delivered to the nucleus to initiate a new round of propagation. Could a unique portal vertex be used for initial disassembly or protein VI release and nuclear import during infection and DNA packaging? If a single vertex pops off from the disassembled HAdV-C5 capsids, how would the genome become available to detection by cytosolic PRRs in some cell types?
Clearly, cell type-specific trafficking influences the interaction with PRRs and therefore may provide an opportunity for vector optimization. We all know that cells differ in their morphology and functions, and it would be surprising if they interacted with pathogens in identical manners. A distinct variation on AdV trafficking has been observed in neurons. The existing Open Comments threads will continue to exist for those who do not subscribe to Independent Minds. Due to the sheer scale of this comment community, we are not able to give each post the same level of attention, but we have preserved this area in the interests of open debate.
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