1 Key Laboratory of Medicinal Chemistry Biology, College of Chemistry, Research Center for Analytical Sciences, School of Medicine, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China
2 College of Chemistry and Molecular Sciences, Research Center of Analytical Sciences, Wuhan University,Wuhan 430072, China
Dai-Wen Pang
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Received
Revised
Published Online
2020-10-27
2020-11-18
2020-12-09
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Abstract
Viruses are one of the biggest threats to human health, and the outbreak of viral diseases not only poses a great threat to human health and national security, but also causes great losses to the social economy. Uncovering the mechanisms of virus infection is crucial for preventing the spread of viruses and treating viral diseases. The dynamic process of virus infection in host cells involves intricate interactions between viral components and cellular structures or organelles, but conventional methods lack the ability to acquire dynamic information on individual viruses during the infection process. Single-virus tracking(SVT) technique is a powerful approach for studying the real-time and in-situ dynamics of viral processes in live cells and it plays an increasingly important role in the study of viral infection mechanism. SVT allows researchers to obtain the dynamic information on individual viruses during the infection process, including viral entry, trafficking, and genome release, which is meaningful to study the infection mechanisms on the molecular level. In this article, we first discuss the measurement techniques, viral labeling strategies and data analysis methods for SVT, then summarize a couple of applications of SVT and finally propose the challenges and future possibilities of the SVT technique.
Contents
1 Introduction
2 Single-virus tracking technique
2.1 Measurement techniques
2.2 Viral labeling strategies
2.3 Data acquisition
3 Applications of single-virus tracking in virological research
On October 5, 2020, the Nobel Prize in Physiology or Medicine was awarded to Harvey J Alter, Michael Houghton and Charles M Rice, three scientists who discovered hepatitis C virus, for their decisive contribution to the fight against blood-borne hepatitis and the reduction of the incidence of cirrhosis and liver cancer. However, at this time, people around the world are fighting a deadly battle against another virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 is highly infectious, pathogenic and lethal. It took only two months from the outbreak of COVID-19 in December 2019 to sweep the world[1,2]. According to who statistics, as of October 25, 2020, there were more than 42.51 million confirmed cases of COVID-19 infection worldwide, resulting in at least 1.14 million deaths, but the actual number of infections and deaths may be far more than that. In addition to posing a serious threat to the lives of people around the world, the COVID-19 outbreak also caused economic stagnation, pushing nearly 100 million people worldwide into extreme poverty. In addition, AIDS, severe acute respiratory syndrome (SARS), hand, foot and mouth disease, Ebola hemorrhagic fever, influenza and other diseases that seriously threaten human health, social economy and national security are all caused by viruses[3]. Therefore, a comprehensive and in-depth analysis of the infection process of the virus is essential to understand its pathogenic mechanism, establish diagnosis and treatment methods, and formulate prevention and control measures!
Viruses have a wide variety of forms, mainly spherical (e.g., HIV, fig. 1A), rod-shaped (e.g., baculovirus, fig. 1B), filamentous (e.g., Ebola virus, fig. 1C), rhabdoidal (e.g.rabies virus, figs. 1D), brick-shaped (e.g.Orthopoxvirus;Fig. 1E), coronavirus (e.g., novel coronavirus, fig. 1F), astrovirus (e.g., astrovirus, fig. 1G), icosahedron (e.g., adenovirus, fig. 1H) and the like, typically consisting of a nucleic acid that carries genetic information and a protein capsid that protects the nucleic acid[4][5][6][7,8][9][10][11][12]. According to whether there is an envelope outside the capsid, viruses can be divided into enveloped viruses (such as herpes virus, baculovirus, Ebola virus, rabies virus, poxvirus, new coronavirus, influenza virus, etc.) And non-enveloped viruses (such as astrovirus, adenovirus, etc.). Viruses depend on host cells to proliferate, and the infection process is the integration of a large number of transient molecular events that occur in turn in the process of spatiotemporal dynamic changes, including receptor recognition, endocytosis, intracellular transport, nucleic acid release, replication, packaging and progeny virus release, each of which involves a variety of pathways and complex mechanisms. The diversity and complex infection process of viruses make the study of viral pathogenesis a key scientific problem in biomedical research that needs to be solved urgently and has not yet been well solved. Traditional static detection techniques (such as biochemical experiments and transmission electron microscopy imaging) can obtain the structure of the virus, so as to analyze the infection information of the virus in a specific time, space and average. For example, SARS-CoV-2 virus binds to cell surface receptors through surface spike proteins, which is the first step of viral infection of cells.
Determines the ability of the virus to infect. Researchers found that, unlike SARS virus, SARS-CoV-2 virus has fewer surface spike proteins (50-100 in SARS virus, less than 30 in SARS-CoV-2 virus on average) and can rotate freely on the surface of the virus. Because of this feature, when the virus infects cells, it can freely adjust the orientation of the spike protein to facilitate the binding of cell surface receptors. The analysis of the internal structure of SARS-CoV-2 revealed that its ribonucleoprotein complex was locally ordered, with a large number of ribonucleoproteins arranged in a hexameric "bird's nest" manner close to the viral envelope, while in the center of the virus, they were arranged in a regular tetrahedral manner, which may be conducive to the interaction between ribonuleoproteins and nucleic acids. The structure of SARS-CoV-2 virus was analyzed by high-resolution cryoelectron microscopy to show its "true face", which is the basis for understanding the virus and studying its infection mechanism, and lays the foundation for precise development of vaccines and neutralizing antibodies. However, the process of virus infection is a biological event with changing time, space, structure and activity. It is difficult to obtain the real and detailed information of the dynamic process of virus infection only by relying on static technology, and it is even more difficult to reveal its infection mechanism.
With the development of microscopy, the single virus tracing technique, which is used to study the real-time dynamic changes of heterogeneous systems, has been established and developed rapidly, and has become a powerful means to study the mechanism of virus infection. Single virus tracing technology can not only trace the infection process of multiple viruses in groups, but also analyze the infection behavior of a single virus in cells individually, which has become a popular technology to study the mechanism of virus infection. With the help of this technology, researchers can obtain dynamic information such as viral entry pathway, intracellular transport process, viral nucleic acid release and its interaction with cells during viral infection, which makes up for the shortcomings of static research methods.It is of great significance to obtain more detailed and complex information and truly interpret the spatiotemporal dynamic process of virus infection, which is of great significance to the comprehensive analysis of the mechanism of virus infection and the design of new antiviral drugs.
In this paper, we will review the measurement technology, labeling technology and information acquisition involved in single virus tracing, summarize the achievements of single virus tracing technology in the study of virus infection mechanism, analyze the existing problems in this field, and look forward to the future development trend and research focus.
2 Single virus tracing method
The single virus tracing method needs to trace the fluorescently labeled virus particles by using real-time, in situ and dynamic measurement technology to obtain information and further process and analyze the process of virus infection of cells, and then interpret the mechanism of virus infection. It is important to note that since the research object of single virus tracing is usually "live virus" (that is, intact virus particles), the relevant operation must be carried out in the laboratory of the corresponding biosafety level. Based on the research work of our group in the past 15 years, this paper will introduce and review the single virus tracing methods from three aspects: measurement technology, labeling technology and information acquisition.
2.1 Measurement Technique
Early researchers mainly used transmission electron microscopy and biochemical experiments to study the mechanism of virus infection in cells, but this traditional method can only obtain the static information of virus infection from cells, and can not accurately analyze the dynamic biological events in the process of viral infection in living cells in real time. With the development of fluorescence microscopy, single particle and single molecule techniques have been developed vigorously in the field of life science research, and many basic biological problems have been solved successfully.It has greatly improved the research level of protein folding, biomolecular recognition, cell signal transduction and so on, and has become a very powerful tool for biological research[13⇓~15]. Among them, single-virus tracking (SVT) is a technique that uses fluorescence microscopy to track a single fluorescent-labeled virus in real time, visualize the infection behavior of the virus, and analyze the dynamic interaction between the virus and the host cell, which plays an increasingly important role in the study of the mechanism of virus infection. At present, the single virus tracing technology requires high imaging device and labeling technology, and the detected single virus fluorescence signal needs to be strong enough to meet the needs of fine analysis of single virus dynamic behavior. At the same time, when tracing a single virus, the sample often needs to be excited many times to obtain enough data in real time for statistical analysis of the dynamic behavior of the virus, so the light damage to the virus or host cells is also a limiting factor in the selection of instruments, and the exposure time and excitation light intensity should be minimized when imaging. When tracing virus particles in living cells, the autofluorescence of cells often interferes with the target with weak fluorescence signal, so in order to obtain a high signal-to-noise ratio image, the excitation volume should be reduced as much as possible to reduce background absorption and reduce background fluorescence. Therefore, the imaging system generally used for single virus tracing should have the characteristics of high detector sensitivity, fast imaging speed and small excitation volume. At present, the microscopic devices used in the field of single virus tracing mainly include inverted fluorescence microscope, total internal reflection microscope and confocal microscope. For example, the inverted fluorescence microscope has the advantages of large imaging depth and easy operation, but the imaging background noise is large and the interference of cell autofluorescence is serious, so it is not suitable for single virus tracing with low fluorescence labeling signal. Total internal reflection microscopy uses evanescent waves generated by total internal reflection to excite fluorescent samples, which can greatly eliminate the interference of potential fluorescence from non-focal planes and has excellent signal-to-noise ratio and contrast, but it is usually only suitable for imaging fluorescent samples within 200 nm from the interface. Confocal microscopy is a technology that uses the conjugate focusing of the light source pinhole and the detection pinhole relative to the focal plane of the objective lens to image the optical section of the sample, which can effectively remove the stray light outside the focal plane to increase the signal-to-noise ratio and vertical resolution of the image, but its equipment is generally expensive. The above three common microscopic measurement techniques are widely used in the field of single virus tracing, and researchers can choose the most appropriate measurement technique according to the object and purpose of the study.
2.2 Marking Technology
A prerequisite for the implementation of single-virus tracing is the fluorescent labeling of multiple specific components of the virus particles under conditions that do not affect the function of the virus as much as possible. Because of the small size and complex structure of virus particles, mild and efficient labeling is often the key to achieve single virus tracing. In the field of single virus tracing, fluorophores are often used to label viruses, including organic fluorescent dyes, fluorescent proteins and quantum dots. Researchers mainly use the biological method of fusion expression of fluorescent protein and target components and the chemical method of direct or indirect coupling of organic fluorescent dyes or quantum dots with viral components for fluorescent labeling.Each fluorophore has its own advantages and disadvantages, and researchers can make a balanced selection according to the specific requirements of single virus tracing experiments.
2.2.1 Organic fluorescent dye
Organic fluorescent dyes are very small and diverse in size, and can be easily bound to labeled objects by covalent or non-covalent means. They have a variety of uses in the field of fluorescence imaging and have been proved to be indispensable in the fluorescent labeling of biological systems. The fluorescence properties of organic fluorescent dyes are determined by the fluorophore structure and chemical environment, and can be fine-tuned by fine design. In addition to their small size, most organic fluorescent dyes have good photophysical properties, a variety of alternative reactive groups that can be used in various labeling strategies, and a wide range of spectral options. Organic fluorescent dyes are the earliest fluorescent markers used for virus labeling and tracing. As early as 2001, Seisenberger et al described in detail the infection pathway of adeno-associated virus in living cells by tracing the adeno-associated virus labeled by a single Cy5 dye molecule. This report first introduced the single virus tracing technology into virus research and attracted great attention[16]. Subsequently, Zhuang et al. Used fat-soluble dye DiD to label viruses, studied the infection process and endocytosis mechanism of single influenza virus, and revealed the dynamic infection process of influenza virus and the endocytosis process of influenza virus mediated by clathrin[17,18]. However, organic fluorescent dyes have the disadvantages of low fluorescence brightness and poor photostability, which are not conducive to long-term real-time tracking and limit their further application in the field of single virus tracking.
2.2.2 Fluorescent protein
Fluorescent protein was first discovered in jellyfish by Japanese scientists Shimomura in the 1960s. After more than half a century of development, it has been widely used in the field of biology. At present, fluorescent proteins have been derived into a variety of colors, and the spectrum covers almost the entire visible region, thus greatly expanding the application of fluorescent proteins in polychromatic imaging. Especially in the past decade, photoconversion and photoactivation of fluorescent proteins have been skillfully used in the field of high-resolution imaging, thus achieving optical resolution of tens of nanometers, breaking through the limitation of optical diffraction limit[19,20]. Molecular markers based on fluorescent proteins have become a common means to dynamically and visually study the process of virus infection, because researchers can mark the relevant cellular structural components and viral components, especially the specific internal components of the virus, through conventional genetic manipulation. For example, Miyauchi et al. Used green fluorescent protein (GFP) to label the internal structure of human immunodeficiency virus (HIV), revealing that HIV enters the host cell through receptor-mediated endocytosis, and the fusion of virus and endosome occurs in the cell, thus realizing the release of genome[21]. However, the fluorescence intensity of fluorescent protein after labeling is not only related to the brightness of the molecule itself, but also to its number. For GFP and other fluorescent proteins, fluorescence can only be observed after the polypeptide chain is folded and the chromophore is mature, and its expression level is also related to many factors, such as transcription and transfection efficiency, protein stability and folding[22]. In fact, the light intensity of a single fluorescent protein is very weak and the light stability is poor, so it often needs multiple fluorescent protein molecules to label the target at the same time to meet the needs of single virus tracing, which may affect the structure and function of the virus. Fluorescent proteins are also prone to photobleaching, which is difficult to meet the needs of long-term dynamic tracing. Furthermore, the operation of fusing the fluorescent protein with the target component is relatively cumbersome.
2.2.3 Quantum dot
The use of fluorescent nanomaterials such as quantum dots (QDs) to study the dynamic process of virus infection in cells can overcome the shortcomings of fluorescent protein and organic fluorescent dye labeling technology, and greatly promote the study of single virus tracing. Quantum dots (QDs) are a class of semiconductor fluorescent nanocrystals with unique properties such as high brightness (due to strong absorption and high fluorescence quantum yield), good stability (photobleaching resistance), tunable fluorescence emission wavelength, monochromatic excitation and multicolor emission, which are ideal labeling materials for living body tracing and highly sensitive detection of biomolecules[23⇓~25]. In the field of single virus tracing, the high brightness of QDs (10-100 times higher than that of organic fluorescent dyes or fluorescent proteins) improves the detection sensitivity and can obtain high-contrast images. In addition, the excellent photostability of QDs (100-1000 times higher than that of organic fluorescent dyes or fluorescent proteins) makes it possible to trace a single virus for several hours at high time resolution[26]. Based on the above advantages, the single virus tracing technology based on quantum dot labeling has made great progress in the study of virus infection mechanism.For example, our group systematically studied the process of influenza virus transport, acidification and membrane fusion in living cells by using quantum dot single virus tracing technology, and analyzed the transport mechanism of influenza virus in different stages of infection[27⇓⇓~30]. Cui et al. Used quantum dots to label HIV virus and studied the infection process of HIV virus in human macrophages, and carried out real-time dynamic tracing and three-dimensional localization analysis of HIV provirus in latent infected cells integrated with HIV provirus DNA[31,32]. However, quantum dots and other fluorescent nanomaterials also have some shortcomings, such as their properties are difficult to control accurately, and their large size can easily affect the structure and biological activity of organisms.
In addition, the marking strategy has a great impact on the marking technology. How to establish a mild, efficient, specific, fixed-point and quantitative in situ labeling method, and minimize the impact on virus activity, so as to obtain a real and reliable measurement signal is one of the key issues to be solved in the field of single virus tracing. Therefore, there is an urgent need to develop specific and in situ labeling methods for different viral structures and components, and to achieve site-specific quantitative labeling of specific components, so as to accurately interpret the dynamic process of viral infection of host cells.
2.3 Information Acquisition
After obtaining the sequence images of the fluorescent-labeled virus movement by the microscopic imaging device, it is necessary to extract the information of the virus particle movement contained in the images to obtain the information related to the virus infection process, such as speed, trajectory, interaction, etc. (Figure 2). The acquisition of relevant information mainly includes the localization of virus particles, trajectory reconstruction, trajectory analysis and interaction analysis between virus or virus components and host cell components.
2.3.1 Viral particle localization
The first condition for reconstructing the trajectory of a single virus particle is to accurately determine the location of the virus particle. Under the ordinary optical microscope, because the resolution is limited by the diffraction limit, the light spot width formed by the point particles on the image is related to the wavelength (λ) and the numerical aperture (NA), which is approximately equal to λ/NA. For example, for visible light, the spot diameter of a punctiform particle is about 200 nm. Therefore, a specific method is needed to determine the central position of the light spot to meet the needs of single virus tracing experiments. At present, several high-precision positioning algorithms have been developed. They are mostly located based on the fact that the light intensity distribution of a Point light source under a microscope conforms to the Point spread function (PSF) (Equation 1).
$PSF(r)=(\frac{2J_{1}(\frac{2\pi NAr}{λ})}{r})^2$
Where R is the distance to the center of the PSF, the J1 is a first-order Bessel function, NA is the numerical aperture, and λ is the wavelength. Firstly, the location of virus particles can be recovered with high accuracy by deconvolution, which is achieved by fitting the image of virus particles with PSF distribution formula. Furthermore, because the light intensity distribution of a single fluorescent virus particle conforms to a two-dimensional Gaussian function, the image of the single particle can be fitted by a two-dimensional Gaussian formula to realize the high-precision positioning of the single virus particle[34]. Although the two-dimensional Gaussian fitting method has the advantage of high positioning accuracy, it also has the disadvantages of slow calculation speed and cumbersome steps. In addition, the centroid method is also an algorithm to locate the facula, which has the advantages of fast calculation, no complex fitting and iteration process, and no need to consider the theoretical model of facula distribution, but the algorithm is seriously disturbed by the background, which is easy to produce calculation deviation and low positioning accuracy. In order to overcome the shortcomings of the above two algorithms in spot location, especially in three-dimensional location, our research group developed a new three-dimensional tracking algorithm based on radial symmetry.The algorithm has the advantages of high positioning accuracy (the positioning accuracy can reach ~ 5 nm) and fast calculation speed (about two orders of magnitude faster than the Gaussian fitting method), and is particularly suitable for the application of fast and high-precision three-dimensional single virus tracing[35].
2.3.2 Trajectory reconstruction
Trajectory reconstruction is to connect the positions of the same virus particle in each frame of the picture. Although many virus particles can be accurately located in video images, the movement of viruses in living cells is very complex.For example, in the process of tracing, spot disappearance, spot merging and spot splitting will occur between the frames of the collected spot images, so the steps of reconstructing the trajectory of the virus are very complex. At low virus particle concentrations, the nearest neighbor algorithm is often used to identify the location of the same particle from different frames. For the high concentration of virus particles, the occurrence of interactions between particles greatly increases the difficulty of facula connection, and researchers usually use manual reconstruction methods to solve the above problems. However, manual reconstruction methods are time consuming and lack operational objectivity. Researchers often need to reconstruct the trajectory through effective automated algorithms, and then verify the final trajectory through manual methods. Automated reconstruction algorithms usually use criteria such as spot size, distance, and intensity to connect the same spots, and they must also have the ability to discard uncertain trajectories. A series of algorithms have been developed to solve the above problems, such as algorithms to solve the flicker of light spots, automatic detection and tracing. It is worth mentioning that Jaqaman et al. Have established a very powerful multi-hypothesis tracing algorithm for the problems of particle crossing, fusion, splitting, interaction, light scintillation and light bleaching, which can be effectively used for the study of high-concentration single particle motion behavior[36]. The algorithm first connects the particle positions between successive frames, and then connects the resulting trajectory segments into a complete trajectory. Both steps are formulated as a global combinatorial optimization problem, the solution of which determines the most likely set of particle trajectories in the entire video image.
2.3.3 Trajectory Analysis
Once the trajectory of the virus is collected, a series of motion parameters related to virus infection can be quantitatively obtained. The dependence of Mean square displacement (MSD) on time interval (Δt) is the main method to determine the movement mode of virus particles, from which the diffusion coefficient and fitting speed of virus movement can be further obtained. Here, we briefly introduce the dependence of MSD on time interval (Equation 2), and the rules and methods used for trajectory analysis.
Where R (Δt) is the particle position at the Δt time point, Δt is the time interval, N is the total number of frames, and n and I are integers.
By analyzing the relationship curve between MSD and time interval, the motion trajectory of a single particle can be generally divided into the following four motion modes: 1) directed motion with diffusion; 2) normal diffusion; 3) anomalous diffusion; and 4) corralled diffusion. Through the corresponding trajectory analysis and data fitting, we can analyze the movement behavior of the virus in the cell and the interaction with the host cell components, so as to interpret the dynamic mechanism of virus infection.
3 Progress of single virus tracing in the field of virus research
Viruses infect host cells by entering the cell, transporting to a specific location in the cell, releasing the genome, replicating progeny viral components, and assembling and releasing the progeny virus. The progeny virus can continue to infect new host cells and enter the next replication cycle. For most viruses, it takes only 4 to 16 hours to complete a round of infection. In such a short period of time, a series of fine and complex interactions between viruses and different structures of host cells occur, and single virus tracing technology is to study these interactions.
3.1 Viral entry
In order to infect a host cell, the virus must first cross the plasma membrane barrier and enter the cell interior. According to the different forms of plasma membrane participation, the mode of virus entry into cells can be generally divided into two types: fusion with the plasma membrane and endocytosis. In addition to HIV and pseudorabies virus, most viruses enter cells through endocytosis, such as dengue virus, hepatitis B virus and vaccinia virus[37][38⇓~40]. The development of single virus tracing technology provides a powerful tool for understanding the mechanism of virus entry. Zhuang et al. Found through single virus tracing that after influenza A virus (IAV) binds to cells, very few of them will use the existing clathrin coating pits on the plasma membrane to enter cells, and most of them will recruit clathrin to form new coating pits at their binding sites, thus entering cells[41]. Subsequently, our research group used quantum dot-labeled single virus tracing technology to carry out three-color tracing research and found that IAV recruited clathrin after binding to the plasma membrane, and immediately recruited the initiating protein that could split the plasma membrane after the recruitment reached the plateau.Once recruited, prion immediately dissociates from the virus-enveloping vesicle, followed by clathrin, and the virus encapsulated in the vesicle is rapidly transported into the cell within seconds (Figure 3)[29]. However, not all viruses that recruit clathrin and dynamin can enter cells[29]. Roughly speaking, only about 10% of IAV viruses can bind to the surface of host cells, and less than 20% of the viruses that bind to cells can start to recruit clathrins, and only about 30% of them can recruit enough related proteins to enter cells. It can be seen that only a few of the 1000 viruses can successfully enter cells in the end. In addition, the entry of viruses into cells was not synchronized, and some viruses bound to the cell membrane at the same time could enter cells within 1 to 2 minutes, while others needed 30 minutes or more to enter cells. For IAV and Japanese encephalitis virus, most of the virions entered the cell within 30 min after binding to the cell membrane[27,42].
3.2 Intracellular transport of virus
Viruses entering the cell by endocytosis are entrapped in endocytotic vesicles and transported along the cytoskeleton to or near the nucleus. Rab5 and Rab7 are proteins responsible for regulating the transport and maturation of vesicles. As the vesicles mature, the pH value in the vesicle cavity gradually decreases, providing a suitable environment for different viruses to release their genomes[43][37]. Viruses that enter cells via endocytosis rely on Rab5-regulated endosomes for transport, and viruses that require lower pH to release their genomes also rely on Rab7-regulated endosomes for further transport[44⇓~46][38,47]. In addition, the viral transport process is mainly directed along the microfilament and microtubule cytoskeleton[27,48,49]. Taking IAV as an example, most of the virus entering the cell is wrapped in the early endosome containing Rab5, transported along the microfilament for a short distance under the drive of myosin VI, and then transported along the microtubule to the nucleus under the drive of dynein (Fig.Rab5-containing endosomes recruit Rab7 or fuse with Rab7-containing endosomes, so that the virus is encapsulated in a transitional endosome that contains both Rab5 and Rab7. The transitional endosome further matures into a late endosome that contains only Rab7 and has a lower pH, providing acidic conditions for the release of the IAV genome[27,43,50⇓ ~52]. In addition to this endocytic transport pathway, which is widely used by different viruses, IAV also uses autophagy to transport, and about 20% of the viruses entering the cell are encapsulated in autophagosomes without Rab5 and transported along microtubules to the nucleus (Fig. 5)[53]. Dengue virus also uses autophagosomes for transport during infection of cells[54]. Considering that many kinds of viruses have Rab5-independent transport, it is speculated that autophagy is most likely a transport pathway widely used by different viruses. In addition, unlike the ultra-low efficiency of cell entry, the efficiency of virus transport in the cell is extremely high, and almost all viruses entering the cell can eventually be transported to the vicinity of the nucleus.
3.3 Virus Release Genome
After the virus reaches the interior of the cell, it must release the genome from the dense package into the cytoplasm or nucleus before it can replicate. Endocytic viruses mostly release their genomes by fusion of the envelope with the endosomal membrane at low pH. Based on this, there are two kinds of methods to study the release of viral nucleic acid by single virus tracing: monitoring the fusion of viral envelope and endosomal membrane and monitoring the separation of viral nucleic acid from envelope[55]. Zhuang et al. Labeled the virus envelope with a relatively high concentration of DiD dye, and monitored the process of fusion of dengue virus with the endosome membrane by using the dye fluorescence self-quenching at high concentration and self-quenching at low concentration. It was found that 80% of the virus fused with the late endosome containing only Rab7, 15% with the transitional endosome containing Rab5 and Rab7, and 5% with the early endobody containing only Rab5[38]. However, traditional virological studies have shown that flaviviruses such as dengue fever do not rely on Rab7 protein to infect host cells[56,57]. Therefore, a large number of studies are needed to further verify. In this study, nucleic acid dye, fluorescent protein and quantum dots were used to label the nucleic acid, capsid and envelope of baculovirus, respectively. By monitoring the separation of nucleic acid dye, fluorescent protein and quantum dot signals, the process of baculovirus releasing nucleocapsid into insect nucleus was observed[58]. Cui et al. labeled the nucleic acid and envelope of IAV with quantum dots of three colors respectively, and observed the separation of quantum dots of different colors near the nucleus, and found that about 30% of the viruses entering the cell released their genomes near the nucleus (Fig. 6)[59]. From the above results, we can see that only about one in a thousand viruses can enter cells and successfully release their genomes, which provides a lot of room for drug treatment.
3.4 Virus assembly and exocytosis
After releasing the genome from the interior, the virus also needs to replicate daughter nucleic acids, synthesize daughter proteins and assemble key components into daughter virions, which are released outside the cell. Due to the lack of corresponding labeling methods, single virus tracing technology faces great challenges in studying these processes, but some important progress has been made in the transport and assembly kinetics of key components of progeny viruses and their mechanisms of action. With the help of gene editing technology, Chen et al. visualized the translation process of HIV virus RNA, and found that about half of the RNA in the cytoplasm would be translated, while the other half would not be translated. Both translated and untranslated RNA would be actively transported to the plasma membrane, but only untranslated RNA would be wrapped by Gag protein of the virus and assembled into progeny virus (Fig. 7)[60]. After the assembly of progeny HIV, Gag protein will recruit ESCRT complex and VPS4 protein of host cells to promote the separation of viral envelope from plasma membrane and release the virus out of the cell[61]. Although the virion contains a large amount of actin, actin filaments (microfilaments) are not involved in the assembly of HIV virus on the plasma membrane[62]. In addition, the tracing study of pseudorabies virus found that filial capsid protein was synthesized in the cytoplasm and then entered the nucleus to assemble with filial DNA to form filial virus nucleocapsid (Fig. 8), and then the nucleocapsid and the cell membrane system containing viral envelope protein were assembled in the Golgi apparatus to form filial virus.The progeny virus was encapsulated by acidic secretory vesicles containing Rab6, Rab8 and Rab11, transported along the microtubule skeleton to the plasma membrane, and finally exited the cell through the exocytosis pathway near the anchoring site of microtubules on the plasma membrane[63, 64,65]. Tracing studies of IAV have shown that the virus ribonucleoprotein complexes (vRNP) of progeny viruses bind to Rab11 protein of the host through PB2 protein near the center of microtubule organization.Viral inclusions without membrane structure are formed at the exit of endoplasmic reticulum, which fuse and divide with each other to promote the interaction of different vRNPs of viruses, and finally reach the cytoplasmic membrane under the transport of microtubule and microfilament transport system to assemble progeny viruses[66, 67,68].
In conclusion, single virus tracing is playing an increasingly important role in virology research.
4 Existing problems and countermeasures
Although great progress has been made in the study of viruses, there are still some problems to be overcome as a developing and emerging technology.
4.1 Marking Technology
In general, the labeling technology involves two aspects, namely the marker and the labeling strategy. As can be seen from the foregoing discussion, a fluorescent marker for single virus tracing must have excellent properties, specifically, the marker is required to have a strong tracing signal (fluorescence), high stability, as little non-specific adsorption as possible, good biocompatibility, as small a size as possible, and the like. The fluorescence intensity of organic fluorescent dyes and fluorescent proteins is weak, the photochemical stability of most of them is not very good, and they are easy to photobleach. Some organic fluorescent dyes have strong non-specific adsorption, and the volume of fluorescent proteins is large. Markers with poor photochemical stability often produce free radicals when illuminated, which may damage the normal infection process and even the structure of the virus. At the same time, quantum dots as virus markers also have some problems, such as large size, poor biocompatibility, non-specific adsorption and so on.
The countermeasures to solve the above problems should be considered from two aspects. On the one hand, from the point of view of design and synthesis control, the structure of the markers should be further improved to improve the performance of the markers currently used. On the other hand, new markers with better performance are sought and discovered. For example, multifunctional near-infrared fluorescent quantum dots with ultra-small size (1.5 ~ 1.8 nm), biocompatibility and high fluorescence quantum yield can be synthesized by the quasi-biological synthesis method proposed by our group[69,70].
With excellent markers, it is necessary to establish specific, efficient, fixed-point and quantitative in situ labeling methods according to the structure and dynamic infection process characteristics of the virus, and to minimize the impact on the activity of the virus, so as to obtain a real, reliable and highly sensitive measurement signal. At present, the conventional labeling methods of viral components mainly include: (1) direct use of the differential affinity of labels with specific viral components; (2) that label is directly couple with a specific component of the virus through a biochemical reaction; And (3) fuse and expressing that fluorescent protein and the specific protein of the virus. However, these methods have some limitations, for example, affinity labeling is easily affected by interfering substances and matrices in complex systems, resulting in low binding capacity and specificity, and the binding amount is not easy to control. Biochemical labeling methods are difficult to achieve quantitative control, and the combination of a large number of markers can easily destroy the structure and function of the virus, affecting its activity and normal infection process. The labeled components of fluorescent proteins are limited, and the large expression of fluorescent proteins will also affect the infectious activity of the virus. In addition, in order to accurately and detailedly obtain the multiple information of the whole process of virus infection, different structures and components such as the envelope, the capsid, the nucleic acid and the like of the virus need to be respectively labeled at the same time, but the existing virus double labeling or multiple labeling method generally has the problems of complexity and low efficiency. Therefore, viral markers are still the key to restrict the study of its infection process, and they are also the problems to be solved urgently.
In order to solve these problems, there is an urgent need to develop mild, specific and efficient in situ labeling methods for different structures and components of viruses, and to achieve site-specific quantitative labeling of specific components to ensure their natural infectivity. The possible strategies are the integration of living cell synthesis and in situ labeling, the integration of inorganic and biological systems, and the integration of intracellular biochemical reaction pathways and gene manipulation strategies. For example, through careful design, our team has enabled living cells to automatically synthesize quantum dots and directly label the microvesicles produced by them in situ, thus achieving a labeling efficiency of up to 94% for cell-derived microvesicles with very fragile structures[71]. Of course, this is only the beginning stage of this kind of strategy, but its prospects are very promising. In addition, because the structure of the virus and the key components that interact with the host cell are constantly changing during the infection process, it is very challenging to develop dynamic labeling techniques to adapt to it.
4.2 Measurement Technique
In recent years, the rapid development of microscopy, especially the emergence of high-speed fluorescence microscopy such as total internal reflection fluorescence microscopy and rotating disk confocal microscopy, has promoted the rapid development of single virus tracing research. Especially with the development of super-resolution imaging technology, the super-resolution living cell imaging technology based on rotating disk confocal microscope has emerged, which makes it possible to trace single virus with fast, super-resolution, long-term and deep imaging. However, how to extract and accurately convert the instantaneous, dynamic and small probability event information of virus infection in complex host cells, and develop new strategies such as in situ conversion and amplification of weak signals and dynamic multivariate signal integration.Through the processing and statistical analysis of a large amount of information to correctly understand and interpret the mechanism of virus infection, there are still some bottlenecks, which need to be broken through systematically.
5 Conclusion and prospect
To sum up, the process of viral infection is very complex. With the change of time and space, the structure and biological activity of the virus, as well as the interaction between the virus or its components and the host cell components, are also changing. Although gratifying progress has been made in the research of single virus tracing, and some applications have been made in virology, and even some new discoveries have been made, the method itself needs to be further improved, especially the development of mild and efficient dynamic labeling technology and rapid acquisition and analysis technology of massive data.
As a powerful tool, single virus tracing should be used to study and interpret the infection mechanism of important viruses such as influenza virus, HIV, COVID-19, cell-to-cell transmission mechanism, in vivo infection and pathogenesis as far as possible.It lays the necessary foundation for the establishment of diagnosis and treatment programs, drug research and development, and the formulation of prevention and control measures. It is convenient to grasp the basic knowledge of virology more intuitively and firmly by choosing the frequently infected viruses such as influenza virus as the object, studying and recording the whole dynamic process of their infection in detail, as a live teaching material for the introduction of virology, and it is also of more universal significance.
Tracing measurement of virus infection process at the level of living body can provide the most authentic and reliable information for understanding the mechanism of virus infection and pathogenicity, but because the situation is more complex and difficult, there is no method that can meet the requirements of three-dimensional and long-term dynamic tracing of viruses in vivo. Crucially, the first step is to develop markers suitable for viral in vivo tracing, and near-infrared II quantum dots may be the most worthy of consideration at present. Near-infrared quantum dots (NIR-QDs), as a marker for virus in vivo tracing, not only have the unique properties mentioned above, such as high brightness, good stability (photobleaching resistance), adjustable fluorescence emission wavelength, monochromatic excitation and multicolor emission, but also have the key property of deep fluorescence penetration for in vivo tracing. Of course, you also need to establish an appropriate tagging strategy. With the ideal marker and the corresponding labeling strategy, the corresponding measurement technology needs to be developed. New techniques and methods of three-dimensional single virus tracing with high spatial and temporal resolution in vivo have begun to attract the attention of researchers.
In a word, with the emergence of new labeling materials such as quantum dots with excellent fluorescence properties and the development of high spatio-temporal resolution measurement technology, the single virus tracing method will play an increasingly important role in virology research and even in the prevention and control of major viral infectious diseases.
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