The Herpesviridae is a large family of DNA viruses that are responsible for various diseases in animals as well as humans. The viruses belong to this family are called Herpesvirus. Herpesviruses are grouped together based on biological characteristics and virion morphology and it can be subdivided into three subfamilies such as alpha, beta and gamma herpesviruses. The family name is derived from the Greek word herpin which refers to latent, which means the period of absent clinical signs between reemerging outbreaks of viral replication leading to symptoms. Nine human herpesviruses are known which cause cold sores (Herpes Simplex Virus (HSV-1)), chicken pox (Varizella Zoster Virus (VZV)), … add here infectious mononucleosis B-Cell Lymphomas and Kaposi’s sarcoma (Mettenleiter et al., 2008; Ryan et al., 2014; Baron S, 1996).
2.5.1. Morphology
All herpesviruses are similar in their morphology. The overall size of the virion ranges from 180-300 nm. The linear double stranded DNA and core proteins are encapsulated by an icosahedral capsid. The herpesvirus capsid has a unique covering of amorphous protein called tegument. The tegument contains viral proteins and enzymes that play a structural role and are required for viral replication upon at early stages in infection of the host cell. Herpes viral genome ranges from 125 kbp to 240 kbp of DNA, and it can code around 75 to 200 viral proteins. Herpesviruses also express their own enzymes necessary for viral DNA synthesis, hence herpesviruses can infect cells in both dividing and quiescent stages (Brown & Newcomb, 2011; Baron S, 1996).
The cell tropism of individual virus varies significantly, HSV has the widest range; it can infect many different animal hosts and replicates in numerous animal and human host cells. Human cytomegalovirus (HCMV) can replicate well only in the limited human cell lines like human foreskin fibroblasts. Pseudorabies virus (PRV) infects mainly swine and other mammals such as cattle, sheep, goat, dog and cat (Mettenleiter et al., 2008; Ryan et al., 2014).
2.5.2. Herpes Virus life cycle
To study the interaction between the nucleopore and the capsid of the herpes virus, it is necessary to understand the life cycle of the viral infection and various stages in detail. The Herpesvirus infection starts by attachment of viral envelope glycoproteins with receptors present at the cellular plasma membrane (Mettenleiter et al., 2008). Some herpesviruses can bind to the heparan sulphate on the cell surface proteoglycans, as a part of initial contact. Not all herpesvirus has this mechanism, a notable exception is the Epstein–Barr virus EBV. In general, the herpes virus that use glycoprotein does not require this heparan sulfate for entry, but it will increase the efficiency of viral entry by concentrating virus on the cell surface so that the appropriate viral ligands for entry receptors can find these receptors (Spear and Longnecker, 2003). There are several virally encoded envelope glycoproteins such as B, H, and L, that play essential roles in penetration (Spear, 1993).
After the initial binding of virions to cells, entry of herpesviruses occurs by fusion of the virion envelope with the cell membrane (Nicola et al., 2003) (Wittels et al., 1991). After the fusion a part of the tegument proteins dissociate from the nucelocapsids, these will modulate the cell favorable for viral replication. For example, the tegument protein UL41 of alpha herpesviruses degrades mRNA of the host cell, which leads to virally induced cell shutoff (Kwong and Frenkel, 1989). Then the entered nucleocapsids are directed towards the nucelopore through active transport on microtubules, (Sodeik et al., 1997) a component of the cytoskeleton found throughout the cytoplasm (Pilhofer, et al., 2011). Then the nucelocapsid docks orientation with one of the vertices of the nucleocapsid adjacent to the nucleopore. The nucleocapsids binds to the nuclear membrane, specifically to the cytoplasmic surface components of the nucleopore complex (NPC). According to Insert Reference, when the tegument proteins are removed by using trypsin, efficiency of the capsid binding was reduced. This implies that other tegument proteins present in the capsid plays some role in nucleocapsid attachment with NPC. The genomic viral DNA is released through the capsid portal into the nucleus through unknown mechanisms (Ojala, et al., 2000). After the viral DNA is ejected, the empty capsids are released into cytoplasm. Transcription of the viral genome occurs in a cascade like fashion resulting in sequential expression of immediate-early, early, and late viral mRNAs. Viral DNA replication occurs in a rolling circle fashion producing high–molecular-weight DNA concatemers. The concatemeric DNA, a long continuous DNA molecule repeating the viral genomic sequence, will be cleaved into unit lengths and encapsidated into newly created capsids (Enquist et al., 1999; Roizman, 2001).
Mature nucleocapsids are too large to leave the nucleus through nucleopores. Instead they bud through both nuclear membranes by a unique envelopment-de-envelopment process that is called primary envelopment (Darlington and Moss, 1968). The main goal of the primary envelopment is to transport the nuclear capsid across the nuclear membrane (Ryckman & Roller, 2004). For this process two virally encoded proteins are crucial. These are the products of the UL31 and UL34 genes of HSV1. In the absence of either protein, primary envelopment will be inhibited, and capsids will be accumulated in the nucleus (Chang et al., 1997; Roller et al., 2000).
Figure 1:. Life cycle of Herpesvirus (Mettenleiter, 2004).
After the formation of primary envelope, the primary envelope fuses to the outer nuclear membrane and the capsids are released into cytosol for further maturation. The secondary envelopment process takes place near the golgi apparatus (Mettenleiter et al., 2013). According to Tooze et al., 1993 and Sanchez et al., 2000, the final envelope is derived from early endosomes or the endoplasmic reticulum-Golgi-intermediate compartment. It is still unclear how tegumented capsids are directed to the envelopment site and how viral glycoproteins are assembled there. It has been proposed, that conserved virion tegument component, the UL11 protein, and a conserved viral glycoprotein, gM, are involved in these processes (Mettenleiter et al., 2008). UL11 may influence secondary envelopment by directing tegument components and associated capsids to the budding site, whereas gM is responsible for accumulating envelope glycoproteins at this location. The net result of the secondary envelopment process is an enveloped virion within a secretory vesicle. The vesicle is transported to the plasma membrane, where vesicle and plasma membrane fuse and mature virions are released from the infected cell. Viral components involved in vesicle transport and fusion are UL20 and gK protein (Avitabile et al., 1994; Baines et al., 1991; Foster and Kousoulas, 1999; Fuchs et al., 1997a).
In this project, the two herpesviruses Human cytomegalovirus and Herpes Simplex virus-1 will be used to study the dynamics of interaction between nucleocapsid of the virus and nucleopore of the nucleus in the host cell.
2.6. Herpes simplex virus – 1 (HSV-1)
The herpes simplex virus is categorized under the group alpha herpes virus. HSV-1 are best known for the infections which cause recurrent vesicles and ulcer in the area of skin as well as mucous membranes. HSV-1 is associate with the disease that cause above the waist. It is also called facial herpes (Baron, 1996).
2.6.1. Structure
The HSV-1 is an enveloped virus with diameter of 220 nm. The structure of HSV-1 is similar to the other animal herpesvirus. The HSV-1 has a large double standard DNA. The DNA is enclosed by an icosahedral capsid. The capsid is covered by an envelope made up of lipid bilayer (Mettenleiter, et al., 2006). The HSV-1 has 74 genes which are responsible for development of capsid, tegument proteins and envelope of the virus. These genes are also involved in modulating the replication activity and the infectivity of the virus (McGeoch, et al., 2006). The structure of the capsid portal vertex is recently explored by McElwee, et al., 2018.
2.6.2. Structure of Herpesvirus capsid portal-vertex
The icosahedral capsid of the HSV-1 has a portal at a unique five-fold vertex. The portal is the molecular motor for virus to inject DNA into the nucleus though nucleopore complex during the infection and this portal is the entry point of viral genome into the capsid during the morphogenesis. Due to the small size of the tail like portal-vertex associated tegument (PVAT) and dense layer of tegument between nucleocapsid and the virus envelope, it is difficult to visualize the vertex (McElwee et al., 2018). The capsomers in icosahedral capsid of herpes virus in made up of hexamers and pentamers of a capsid protein pUL19 (VP5) (Cardone et al., 2012).
The 3D reconstruction of cryo electron microscopy has revealed that usual pUL19 is replaced by a unique fivefold symmetric assembly and displays five well defined coiled-coil motifs, each made up of two -helices arranged perpendicular to the capsid surface about the fivefold symmetry. The resulting data of cryoEM shows that the PUL25 C terminal were positioned differently to those at penton-vertices, giving rise to a small tail like assembly, crowns the unique five-fold vertex. In addition, strong density was also detected at the portal-vertex, that were interpreted as DNA. This DNA is packaged and ready for release through the nucleopore (figure 2). The mechanism that triggers the opening and closing of this portal is still not clear (McElwee, et al., 2018).
Figure 2: Structure of HSV 1 capsid portal. (a) Resulting image from cryoEM of HSV-1 capsid and white arrow pointing the portal-vertex with packaged DNA. (b) Sharpened density map of image (a), the packaged DNA is not seen. (c,d) The close-up view of portal highlights the morphology of portal. (e) Segmented density map with three highlighted features, the portal in mauve color, the pentameric portal vertex protein in purple color, and the peri-portal triplex in magenta color. (f) Top view of the portal showing fivefold symmetry axis (McElwee et al., 2018).
2.6.3. Entry into host cell
The entry of the HSV-1 into the host cell is assisted by several glycoproteins present in the viral envelope. The first step of the entry is the binding of glycoproteins to the transmembrane receptor on the host cell surface. After the binding of glycoproteins, the transmembrane receptors of the host cell are pulled inside the cell. This action creates a pore on the cell membrane, by which the nucleocapsid of the HSV-1 will enter inside the cell (Clarke, 2015).
2.6.4. Pathogenesis
2.6.4(a). Acute infection
Initially the HSV-1 infect and replicate in the mucoepithelial cells. Then they will start the productive infection on the contact site. There are various pathological changes occur during acute infection. The pathological changes include the development of multinucleated cells, focal necrosis, development of eosinophilic inclusion bodies and an inflammatory response. Then the virus will spread to local sensory neurons and travels to the sensory ganglia. The latency is developed in the ganglionic neurons.
2.6.4(b). Latent infection
The latent infection of HSV -1 occurs in trigeminal, superior cervical, and vagal nerve ganglia. The latent infection of HSV-1 on the neurons will not leads to cell death, because of the latency established in the non-dividing neurons. The early or late polypeptides are not needed for latent infection. Hence the antiviral drugs aimed for thymidine kinase enzymes will not stop the latent infection. The reactivation of latent viral infection leads to overt disease on some group of patients. The reactivation mechanism of latent infection of HSV-1 is not well defined.
2.7. Human cytomegalovirus
Human cytomegalovirus HCMV also known as human herpesvirus-5 (HHV-5) belongs to genus cytomegalovirus and is a member of the family Herpesviridae. These viruses are frequently found in the salivary gland. HCMV infections are typically unnoticed in healthy people, but can be life-threatening for the immunocompromised patients such as HIV-infected persons, organ transplant recipients, or newborn infants (Ryan et al., 2014). After infection, HCMV remains latent within the body throughout life and can be reactivated at any time. (Melnick, et al., 2011; Geder 1977). HCMV is found in almost all socioeconomic group around the globe (Fulop, et al., 2013). It is one of the major viruses transmitted to the newborn infants during child birth (Britt, 2017).
2.7.1. Structure
The human cytomegalovirus structure consists of an outer lipid bilayer envelope, composed of various viral glycoproteins, followed by the tegument proteins, a proteinaceous matrix. The icosahedral nucleocapsid is formed by seven viral capsid proteins. capsid pentamers and hexamers are formed by the major capsid protein (MCP) UL86. The triplexes are formed by the less abundant minor capsid protein (mCP) UL85 and minor capsid-binding protein (mC-BP) UL46. The small capsid protein UL48.5 is located at hexon tips (Butcher et al., 1998). Finally, UL80 encodes for three additional proteins on co-terminal transcripts that are required for capsid assembly (Loveland et al., 2007). The virion is usually spherical in shape and average size is around 300 nm. The glycoproteins present in the lipid bilayer are glycoprotein B (gB), gH, gL, gM, gN, and gO, are responsible for cell attachment and penetration of virion into host cell. The tegument contains two major types of proteins classified by the function they serve. One type of protein serves a structural role and helps in the assembly and disassembly of the virion during entry. The other type modulates the host cell response to infection (Crough and Khanna,2009).
2.7.2. Transmission
HCMV is transmitted between humans through body fluids such as saliva, tears, urine and blood (Arvin et al., 2007). HCMV transmission on the infants from the mother occurs during child birth and breast feeding. It is known that HCMV can also be transmitted during organ transplantation through blood as well as transplanted organs (Taylor, 2003).
2.7.3. Pathogenesis
HCMV cause disease in immune system suppressed patients, AIDS patients and infants. After infection the virus remains latent in the patient’s body for rest of the life. (Bottieau et al., 2006) (Ryan et al., 2014). The protein UL16 is encoded by CMV, which involved in the immune evasion of NK cell responses (Welte et al., 2003).
2.8. Cell cycle dependency of HCMV
2.8.1 Cell cycle and its regulators
The cell cycle consists of a highly controlled series of steps that promote duplication of the DNA, monitor the DNA for damage, and allows the division into two identical daughter cells once the requirement for all the checkpoint surveillance have been met. The proteins such as cyclin A and cyclin dependent kinase (CDK) serve as the master regulators of each of the cell cycle steps. The cell cycle is divided into four major phases G1, S, G2 and M. In addition, there is a phase of quiescence or resting state called G0, during this phase the cell will be withdrawn from the cell cycle, it occurs between the M and G1 phase. The G1 phase takes place in between mitosis (M) and DNA synthesis (S). Further progress of the cell into G1 phase from G0 phase or M phase, is mediated by expression of the D-type cyclins. Before the DNA synthesis at S phase and after the G1 phase, cyclin E1 synthesis is induced at the transcriptional level (Blow and Dutta, 2005). Cyclin A2 accumulates in S phase and forms an active kinase complex with CDK2. Regulation of cyclin A occurs at both the protein and mRNA levels and the initiation of DNA replication requires the CDK2/cyclin A complex (Machida et al., 2005; Spector, 2015). G2 phase occurs after DNA has been completely replicated and is characterized by the accumulation of proteins required for separation of the chromosomes and mitosis. The mitosis phase is assisted by Cyclin B1 and its association with CDK1 (Millar and Russell, 1992).
2.8.2. Cell cycle in HCMV infected cells
When the cells are infected with HCMV, the cell cycle process is arrested. This cell cycle arrest is due to various factors. The HCMV infection induces the cell cycle progression from G0 phase to G1 phase, but the cell cycle is blocked before the cellular DNA replication, which blocks the cell cycle to proceed further to S phase and expression of cyclins and CDKs are disrupted. It is also studied that HCMV inhibits expression of cyclin D and cyclin B, but promotes the accumulation of cyclin E and B at higher level. The accumulation of cyclin B is due to protein stability, while infection affects the RNA levels of cyclin E and D1. The mechanisms used by HCMV to stop the cell cycle indicates that it is important for viral replication. In accord with this, it was shown that initiation of HCMV gene expression requires the cells to be in G0 or G1 at the time of infection (Salvant et al., 1998; Sanchez et al., 2003; Spector, 2015; Jault et al., 1995; Fortunato et al., 2002).
In S/G2 phase of cell cycle, after virus entry immediate early IE gene transcription is blocked. This block can be relieved by inhibition of cyclin-dependent kinases 1 and 2 (CDK1/2). Cyclin A2 has been identified as the key regulator that recruits CDK1/2 for inhibition of the HCMV lytic cycle (Oduro et al., 2012).
2.8.3. Cyclin A regulation
The hypothesis of responsibility of cyclin A for cell cycle dependency of the HCMV infection is supported by studies using a cyclin A2 over expressing lentivirus-transduced U373 cells and pp150 – RXL mutant HCMV. The pp150 is the major tegument 150 kDa phosphoprotein of HCMV, that binds to cyclin A via function RXL motifs. This binding results in cyclin dependent phosphorylation. This interaction was prevented by alanine substitution of RXL/Cy motif, that allows mutant virus to skip cyclin dependent cell cycle. As a result of the experiment, during the S/G2 phase of cell cycle, the IE (immediate early) gene expression was blocked in cyclin over expressing cell when infected with wildtype HCMV and there is no blockage of IE gene expression when infected with pp150 – RXL mutant HCMV. On other hand the cells at G0/G1 phase, no blockage of IE gene expression was seen during both wild type HCMV and mutated HCMV infection. The result proves that active cyclin A/CDK2 kinase activity was required for inhibition of IE gene expression at S/G2 phase (Bogdanow et al., 2013).
2.8.4. Tegument Protein pp150
The tegument protein pp150 is highly immunogenic and it is produced as a product of UL32 gene. It is the second abundant tegument protein; a virion contains approximately 1500 copies of pp150. It has been studied that this pp150 interacts with performed capsid with its amino terminal of 275 amino acids (Varnum et al., 2004). This amino terminus of pp150 is required for HCMV infection and its essential for productive HCMV replication. It is also essential for maintaining the stability of cytoplasmic capsid and direction of their movement (Tandon et al., 2008). The inhibition of IE gene expression by cyclin A/CDK2 can be understood by observing this tegument protein pp150. It contains multiple canonical CDK-phosphoacceptor sites and putative cyclin A binding motifs (Caffarelli et al., 2013; Bogdanow et al., 2013; Spector, 2015). In this project, we will study pp150 involvement in genome injection of nucelocapsid through NPC.
2.9. Nuclear pore complex
The nuclear pore complex (NPC) is a large assembly of proteins that provides the only portal for exchange of macromolecules in and out of the nucleus. This portal is present in between the inner and outer nuclear membranes. The diffusion of macromolecules is performed either by passive diffusion or by signal mediated process. The NPC’s is the only mediator of nucleocytoplasmic exchange; hence this pathway affects all sorts of cellular physiology that are required for transport. That includes gene expression, growth and proliferation, signaling, cell cycle control, and death (Vasu and Forbes, 2001; Weis, 2003).
2.9.1. Structure
The NPC has an eight-fold symmetrical structure were the inner and outer nuclear membrane join. It has a central transport region that is cylindrical in structure surrounded by cytoplasmic and nuclear rings, perpendicular to the plane of nuclear envelope. There are eight equally spaced spokes that radiate from the inner spoke ring surrounding the central region. Each of the spokes are composed of several struts. These spokes are attached to its neighboring spokes by four coaxial rings, such as inner spoke ring, outer spoke ring or lumenal ring, cytoplasmic ring and nuclear ring. The cytoplasmic and nuclear ring are morphologically different in nature. It has eight cytoplasmic filaments attached to eight cytoplasmic particles, which stick to the cytoplasmic ring. The nuclear basket is formed by eight filaments attached to nuclear ring and it converges to terminal ring forming a basket like structure. The digramatic structural representation of the NPC is shown in figure 3. (Rout & Wente, 1994; Suntharalingam & Wente, 2003).
Figure 3: Schematic diagram of NPC strucutre and its parts (Suntharalingam & Wente, 2003).
2.9.1(a). FG Domains
The NPCs are assembled from copies of proteins called nucleoporins. These Nuceloporin contains non-globular domains rich in FG dipeptide motifs called FG domains. It also has a globular domain which is used for attachment of nucleoporins to the NPC (Lodish, et al., 2008). FG-repeats are small hydrophobic segments made up of 4–48 GLFG, FxFG, SxFG, or PxFG motifs which are separated by spacers of different length. These FG repeats have an important role on mediating the translocation of transport receptor–cargo complexes through the NPC by providing important interaction surfaces for transport factors (Zeitler & Weis, 2004). These segments of FG-nucleoporins form a mass of chains which allow smaller molecules to diffuse through but exclude large hydrophilic macromolecules. The large molecules can able to cross a nuclear pore only if they have a signaling molecule that interacts with a nucleoporin’s FG-repeat segment (Lodish, et al., 2008). FG domain fills the central channel present in the central region of the NPC. It forms the NPC permeability barrier. Apart from the non-globular domain, it also includes an intrinsically disordered region proteins or IDR. About one third of total nucleoporins consists of IDR. IDR does not have definite 3D structure. Apart from the water-soluble proteins, other proteins in IDR form a protein rich phases including hydrogels, liquid droplets or amyloid like aggregates (Schmidt and Görlich, 2016).
2.9.1(b). NUP214
Nucleoporin 214 is a protein encoded by the gene NUP214. This gene is a member of FG repeat containing nucleoporin. These NUP214 are present in cytoplasmic end of nucleopore complex. It is responsible for regulation of proper cell cycle progression and nucleoplasmic transport. Studies have reported that NUPs are constructed by a small number of major structural domains: coiled-coils, FG repeats, α helical solenoids, β-propellers, and zinc fingers. The N-terminal domain (NTD) of the NUP214 can be divided into two parts N-terminal canonical seven-bladed β-propeller domain and it is followed by C-terminal extended peptide segment (CTE). The NTD is followed by coiled coil and FG repeats as shown in Figure 4. (Napetschnig, et al., 2006). The nucleoporin NUP214 involvement in triggering the DNA injection of the herpesvirus nucleocapsid is explained previously by Pasdeloup, et al., 2009. In this project we will develop a stable cell line expressing the long construct of FG repeats, tagged with fluorescent protein mcherry. In this cell line the FG repeats are isolated from the NPC as a phase separated liquid in the cytoplasm. When infected by the herpes virus and new capsids are released, we will use live cell imaging to find out whether this phase separated FG repeats can bind to the capsid and are responsible for genome release.
Figure 4: The Domain structure of NUP214 (Napetschnig, et al., 2006).
2.9.2. Fnction
Various mechanisms of nucleoplasmic exchange occur with the help of NPC. There is transport channel in NPC, though which the transport in and out of the nucleus takes place. The diameter of the transport channel is 9 nm, which was determined by diffusion studies. The exact location of the channel is not clear, it has been hypothesized that these channels are present in between spokes and nuclear envelope or in between central region and spokes. Ions and small molecules can be transported through passive diffusion. The macromolecules which are bigger in size than the diameter of the channel was actively transported via NPC. The transport process is fast, energy dependent, regulated and highly selective. All NPC can be active in both nuclear import and export functions. The NPC also forms a part of extended karyoskeleton and it is attached to nuclear lamina and nuclear lattice (Mehlin, et al., 1992; Rout and Wente, 1994; Suntharalingam & Wente, 2013).
2.9.3. Selective phase model
here are various models that explain the nuclear transport process reviewed by D’Angelo and Hetzer, 2008. The selectivity of NPCs and role of FG repeats in the process, is explained in the selective phase model.
This model states that, the central channel of nuclear pore complex is formed by the FG nucleoporins. The central channel is filled with a gel like semi-liquid phase of FG nucleoporins, which assist in selective transport of macromolecules across the NPC. The transport receptor can easily partition into this FG nucleoporin but the macromolecules above certain size cannot pass through it. This model also explains that the translocation of cargo molecules would be promoted by transport receptors. This can be achieved by increasing the solubility of transport receptors in the central plug. This model explains, how the selective physical barrier made up of semi liquid FG repeats could work (Ribbeck and Görlich, 2001).
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Figure 5: Selective phase model for facilitated transport through NPC. (A) The permeability barrier of central plug in NPC filled with meshwork of FG repeats (light blue circles). (B) A macromolecule with binding sites for FG repeats (dark blue circle). (C) The macromolecule’s binding site interacting with FG repeats, which makes the macromolecule part of the semi liquid phase. Further, the macromolecule will dissolve in the central plug, which allows it to cross the physical barrier (Ribbeck and Görlich, 2001).
2.10. Viral DNA labelling
The Viral DNA labelling is a method to visualize the viral DNA injection into host nucleus by using fluorescent microscopes. The current methods of the viral DNA tracking have some limitations such as low resolution and accuracy to visualize the viral DNA at single particle level (Wang, et al., 2013). In this project, different viral DNA labelling methods for the live cell imaging are compared.
2.10.1. Click chemistry
The click chemistry is a robust method for labelling the specific biomolecules using a substrate or a fluorescent probe. The term click chemistry was coined in 1998 by an American chemist K. Barry Sharpless in 1998. In recent years, the click chemistry has been implemented in the live cell imaging. The molecular probes will specifically bind to the biomolecules in the live cell using the click reaction (Kolb, et al., 2001, Evans, 2007). There are various click reactions are in use. In this project, the click chemistry was used to label the DNA of the HCMV PP150-GFP virus. The fluorophore such as Janelia 549 and Tamra dye are used for labelling the DNA.
2.10.1(a). Alkene and tetrazine inverse-demand Diels-Alder
In this project, the click chemistry reaction is based on the Alkene and tetrazine inverse-demand Diels-Alder reaction. The activated alkene reacts with tetrazine in an inverse-demand Diels-Alder. The invDA reactions do not require a catalyst and it is also known to be compatible is cell media. These properties of the reaction make it desirable for its application on the live cells. Hence, the tetrazines can react with aromatic vinyl compounds, 5-vinyl-2’-deoxyuridine (VdU). VDU is identified as suitable label for DNA (Rieder and uedtke, 2014).
Figure 6: The reaction between VDU and 3,6-di-2-pyridyl-1,2,4,5-tetrazine 5 (py2-Tz)
2.10.1(b). C9C9-TriPPPro-VDU
In this project, the VDU modified by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany) is used. The modified VDU is linked with C9C9 using the linker TriPPPro. The chemical composition of the modified VDU is shown in the figure 7.
Figure 7: Chemical composition of C9C9-TriPPPro-VDU. Modified by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany).
2.10.1(c). Janelia Fluro 549 Py2Tz
In this project, Janelia fluro 549 Py2Tz was synthesized by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany) is one of the probe used to click with VDU C9C9-TriPPPro-VDU. The chemical formula of Janelia 549 Py2Tz is C47H43N11O6 and the molecular weight is 857,932 g/mole. The Py2Tz (3,6-di-2-pyridyl-1,2,4,5-tetrazine 5) present in Janelia Fluro 549 acts a linker to click with VDU bound to viral DNA. The chemical composition of the Janelia Fluro 549 is shown in the figure 8.
Figure 8: Chemical structure of Janelia Fluro 549 Py2Tz. The chemical composition of Py2Tz is marked in red color. Modified by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany).
2.10.1(d). Tamra Py2Tz
The tamra Py2Tz is another fluorophore used in this project for DNA labeling. The chemical formula of tamra Py2Tz is C34H29N7O4 and the molecular weight is 599,651 g/mole. In this project, the linker Py2Tz in the tamra dye will be clicked with VDU bound to the DNA of the virus. The Tamra Py2Tz was synthesized by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany). The chemical structure of tamra Py2Tz is shown in the figure 9.
Figure 9: Chemical structure of Tamra Py2Tz. The chemical composition of Py2Tz is marked in red color. Modified by Vicente Sterrenberg (Prof. Dr. Chris Meir’s lab, University of Hamburg, Germany).
2.10.2. Syto 82 dye
In this project, apart from click chemistry, the cell permeable dye is also used for labelling viral DNA. The Syto 82 orange flourosacent dye is a cell permeable flouroscent dye stains the nucleic acid. The syto 82 dye can diffuse through the cell membrane and stains the nucleic acid. Hence, the Syto dyes are cell permeant and it has net positive charge at neutral pH, they may also stain the mitochondria of the cell. The optimal excitation range of Syto 82 is in between the wavelength of 530 nm to 570 nm. Unlike the other dyes used in click chemistry, the syto dye could not be used to stain cells in growth medium. Hence the growth medium should be replaced by buffer such as Phosphate-buffered saline. In this project the purified HCMV viral DNA is stained by syto 82 orange dye and the efficiency of the staining was compared with other click chemistry dyes (Molecular probes-Thermofisher scientific, 2001).
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