NanomedicineVol. 8, No. 8 EditorialFree AccessTracking viral infection: will quantum dot encapsulation unveil viral mechanisms?Zhenhua Zheng & Hanzhong WangZhenhua ZhengState Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Xiaohongshan No. 44, Wuhan, 430071, ChinaSearch for more papers by this author & Hanzhong Wang* Author for correspondenceState Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Xiaohongshan No. 44, Wuhan, 430071, China. Search for more papers by this authorEmail the corresponding author at wanghz@.wh.iov.cnPublished Online:5 Aug 2013https://doi.org/10.2217/nnm.13.114AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: enveloped virusnonenveloped virusquantum dotvirus labelingvirus trackingTracking viral infection in living cells and organisms will facilitate the development of novel therapeutic targets for the control of viral diseases, as well as the improvement of virus-mediated transgenic technology [1]. Quantum dots (QDs), with the characteristics of brightness, photochemical stability and size-determined emission spectra, have significant potential for use in biomedical fields [2,3]. For instance, viral components labeled with QDs can be used as imaging probes to study the mechanisms underlying viral entry, trafficking and egress. This editorial reviews the progress in QD labeling of viruses and the applications of QDs.Strategies for labeling viruses with QDs▪ Labeling the surfaces of viruses with QDsThe envelope of an enveloped virus is generally derived from the host cell membrane [4]. Thus, QDs can be attached to the membranes of infected cells, resulting in the budded virus being labeled. You et al. used cationic polybrene to neutralize the negative charge on the surfaces of QDs and retroviruses, and combined them with a colloidal composite [5]. On the other hand, positively charged QDs carrying a positively charged surface were incubated with infected cells with negatively charged cell membranes, allowing the membrane of the virus to be labeled with QDs during virus budding [6]. Both of these labeling strategies are based on electrostatic attraction.In addition, a method based on the tight interactions between biotin and streptavidin (SA) have been developed to label the envelopes of several viruses. For example, the envelope of pseudorabies viruses obtained from membranes of infected cells was biotinylated by adding biotin-cap-PE to the cell culture medium. Subsequently, the virus was incubated with SA-conjugated QDs (SA–QDs) to label the membrane of the virus [7]. Similarly, infectious hematopoietic necrosis virus (IHNV) and influenza A virus H9N2 were labeled with EZ-Link® sulfosuccinimidyl 6-(biotinamido) hexanoate (Thermo Scientific, IL, USA) on their surfaces [8,9], followed by SA–QD labeling on their viral envelopes. Apart from the above-mentioned nonspecific approach, a novel, biocompatible, site-specific approach based on the interaction between biotin and SA to label viral envelopes with QDs has also been reported. By expressing a biotin acceptor peptide tag on the cell membrane of lentivirus-infected cells, the envelope of the lentivirus was site-specifically labeled with SA–QD using biotin ligase (BirA) to modify the acceptor peptide tag and introduce a biotin group onto the surface of the virus [10].After site-specific labeling of the surface of the lentivirus with QDs [10], Joo et al. labeled the surface of a nonenveloped virus, adeno-associated virus (AAV), with QDs following a carbodiimide coupling reaction [11]. This strategy may be suitable for labeling all nonenveloped virus particles and virus-like particles (VLPs).▪ Encapsulating QDs into virusesNonenveloped viruses, such as brome mosaic virus and red clover necrotic mosaic virus, can be labeled through viral self-assembly, by which the viral capsid proteins encapsulate nanoparticles, including QDs, into VLPs under certain conditions. Encapsulation of QDs into the capsids of the brome mosaic virus by self-assembly yielded VLPs with a similar size to the native virus [12]. The VP1 proteins of simian vacuolating virus 40 (SV40) can self-assemble into VLPs in vitro, enabling QDs to be encapsulated into SV40 VLPs through self-assembly [13]. These labeled viruses could then be used to visualize the behavior of the VLPs in living cells.We previously reported that QDs in living cells could be encapsulated into the vesicular stomatitis virus glycoprotein pseudotyped lentivirus (PTLV) without modification of the viral surface [14]. QDs were bound to the modified guide RNAs that contained the sequence of the packaging signal of the viral genome and were encapsulated into the virus particles. This approach may be suitable for all viruses that contain the packaging signal in the viral genome.Tracking viral infections using QDsTo date, the majority of studies tracking viral infection in cells using QD-labeled viruses have reproduced the data on viral infection revealed by conventional methods. For instance, QD-containing SV40 VLP was observed to enter cells through caveolin-mediated endocytosis, be transported along microtubules and eventually gathered in the endoplasmic reticulum region [13]. The revealed pathway is in agreement with that of SV40 infection. QD-labeled AAV2 was shown to gain entry mainly through clathrin-dependent endocytosis and, subsequently, the cytoskeleton network aided transport through a variety of endosomes [10]. These findings indicate that the infection routes of QD-labeled viruses are consistent with those of unlabeled wild-type viruses.QD-labeled IHNV entered into the host cell by clathrin-mediated endocytosis and was transported around the cell via the cytoskeleton before it disassociated in the endosomes [8]. This was the first study using a QD-labeled virus to visualize the dynamics of a virus as well as viral endocytosis, and was the first imaging study on the infection mechanism of a rhabdovirus. By analyzing the position and the instantaneous velocity of the QD-labeled influenza virus in a living cell, it was revealed that the infection process could be split into a five-stage transport model [9]. Both of these studies reported new findings, demonstrating the practicability of the QD-labeled viruses.When the envelope of a pseudotyped virus was labeled with QDs in a site-specific manner, it was shown that VSVG-PTLVs do not enter host cells by clathrin- or caveolin-mediated endocytosis, while VSVG-pseudotyped gammaretrovirus was found to enter into cells via clathrin-mediated endocytosis [10]. These findings suggest that the viral membrane may not be the sole factor that determines virus invasion. We previously reported that QD-containing PTLV had a similar efficiency of entering into cells like the wild-type PTLV [14]. However, how the QD-containing PTLV enters cells and whether this is consistent with the findings reported by Joo et al.[10] require further study.The challenge of tracking virus infection by QDsAlthough a growing body of evidence indicates that QD labeling does not affect the biological characteristics of the labeled viruses, tracking viral infection using QD-labeled viruses remains a challenge.Although QDs have been used to track viral infection, whether it affects the cellular intake of biological molecules remains unknown. The behavior of biological molecules may change when they bind to QDs [15]. QDs can increase or decrease the transport efficiency of many toxic molecules [16]. Meanwhile, when a single potassium channel protein was detected by QD-labeled antibodies, the diffusion type changed significantly [17]. In addition, polyvalent crosslinking of the labeled protein with QDs might activate some signaling pathways, thereby affecting the imaging results [15]. Thus, the interaction of QDs with intracellular components may cause unexpected results. As a complex biological entity, being labeled with QDs may, to a certain extent, affect the infection route of a virus.In addition, the particle size of QDs may also influence the infection route of the QD-labeled virus. This is especially true for large-sized QDs. In particular, when QDs are designed to be encapsulated into the virus, their size is essential. Therefore, reducing the size of QDs may facilitate efficient labeling of the candidate virus. In a previous study, we observed that SA–QD, which is not membrane permeable, resulted in retention of transfected QDs in the endoplasmic reticulum, as well as a significant decrease in packaging efficiency [14]. The use of membrane-permeable QDs may improve the packaging efficiency.Cell receptor binding sites located on the surface of a virus are essential for viral attachment and entry into host cells. Therefore, QDs on the surface of a virus may affect the entry efficiency of the virus by blocking the viral ligand from binding to its cellular receptor. This provides one explanation for why the infectivity of QD-labeled IHNV and influenza virus H9N2 decreased compared with that of the wild-type viruses [8,9]. Consequently, to minimize the effect of QDs on the binding of the AAV2 capsid to the cell receptor, an appropriate amount of QDs on the viral capsid should be maintained under certain conditions [10].Multilabeling strategies are neededVirus-mediated membrane fusion is a key step of enveloped virus infection, which can occur on the cell surface or in endosomes [4]. However, labeled fluorescent material is removed from the virus core after membrane fusion, making it impossible to trace viral infection after this step. For example, the lipophilic dye DID on the viral membrane of HIV disappeared after membrane fusion [18]. Encapsulating QDs into the viral capsid makes it possible to track the viral infection route before viral capsid dissociation. To visualize the life cycles of enveloped viruses, including viral entry, transport, genome release, assembly and budding, different viral components should be labeled with different fluorescent materials. Although encapsulated QDs can be used for tracking virus invasion and intracellular transport, to gain more details on the whole life cycle of a virus, a multilabeling strategy, in which more than one viral component is labeled with QDs or other fluorescent material, is needed. For example, if we combine the strategies of ours [14] and Joo et al.[10], QDs could be site-specifically labeled on the surface of the viral membrane and encapsulated into the viral capsid at the same time. This technique will allow us to define the viral mechanisms by which the viral membrane is stripped from the viral capsid and the capsid disappears during release of the viral genome. Recently, it has been reported that the envelope and genomic RNA of influenza A virus (H9N2) were labeled with QDs and SYTO 82, respectively [19]. These studies suggest that dual- or multi-labeling of different viral components is feasible during viral replication in host cells. Combining this with the properties of QDs, multilabeling of viruses with QDs may provide more valuable information for studying viral infection mechanisms.Financial & competing interests disclosureThe authors were supported by grants from the Major State Basic Research Development Program of China (973 Program; 2011CB933600). 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The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download