Dengue virus ns5 antibody

They are non-competitive inhibitors that bind to specific pockets other than the active site [ 81 ]. They act by blocking conformational changes of the enzyme, typically between the initiation and the elongation steps or hindering the binding of the RNA. Their biggest advantage is a high specificity for their allosteric binding pocket, drastically minimizing off-target effects.

However, this is also a weakness as these pockets are more prone to mutations than the active site, leading to the emergence of resistant variants. Moreover, these compounds might have poor pharmacokinetic properties limiting their bioavailability.

Between these steps, a conformational change is thought to occur in order to accommodate the dsRNA. Thus, in principle either the initiation, the elongation and intermediate states in-between can be targeted. The strategy to develop NNI starts with a HTS screening based on structural determination of complexes with fragments originating from the library of compounds to identify potential molecules.

This is followed by rounds of binding biophysical characterization and chemical optimization, using medicinal chemistry principles. Until recently no allosteric pocket for NNI binding had been described for dengue virus RdRp although compounds targeting domains outside of the active site mostly thumb site inhibitors had been reported. A crystallographic compound screening allowed the identification of this pocket.

Then structure guided modifications of the initial hit led to molecules potently inhibiting polymerase activity Table 1. Study of the properties and the inhibition mechanism showed that the compound inhibited the initiation step of the RNA polymerization in all DENV serotypes. Although the compound cannot be used for in vivo studies because of it relatively poor pharmacokinetics properties, this work clearly identified an allosteric pocket from DENV that is essentially shared by the ZIKV RdRp and that can now be targeted using rational design.

Another interesting NNI compound was also identified by HTS based on an activity assay and on optimisation of identified hits [ 97 , 98 ]. UV-cross-linking of the compound showed that it binds the RdRp domain at the edge of the RNA tunnel blocking the template access and interfering in the transition between the open and close conformations of the protein.

In vitro the compound showed promising inhibition activity but its poor pharmacokinetic properties did not permit the development of this compound. Methylation of the RNA cap is essential for virus replication and represents an important drug target [ 26 , 27 , 28 , 99 ]. In the case of West Nile virus, defects in MTase can lead to weakened [ ] or abolished [ ] viral replication capping activity.

Targeting the SAM pocket is an established antimicrobial strategy and several analogues to this ubiquitous molecules exist such as sinefungin [ 16 ], a broad spectrum MTase inhibitor having anti-parasitic activity. This inhibitor has been shown to bind to the SAM pocket of several flaviviruses. Interestingly, its affinity is higher than SAM. However, this compound cannot be used as a drug due to its lack of specificity that will provoke severe side effects since it is not specific to the viral MTase.

Thus, the SAM scaffold was modified by adding substituents at the level of the adenine ring. HTS binding assays identified compounds binding to this pocket and inhibited virus replication, thus validating the strategy. A recent study also showed that a compound can efficiently and specifically inhibit the viral MTase with few cytotoxic effects [ 82 ].

For this, a virtual screening is performed to select possible binders that would later be tested in vitro to assess activity and absence of cytotoxicity. The binding is also characterized by obtaining the crystal structure of the complex with the protein, allowing eventual optimization of the compound [ 82 ]. An interesting strategy was recently used to link compounds obtained through fragment based docking that have been shown to be active as standalone molecules targeting the MTase domain, to improve their potency and specificity [ 96 ].

However, although biochemical activity is improved for the linked compound compared to the initial hits, no activity was observed in the in vitro assays, probably due to poor membrane crossing of the compounds. Recently, an alternative approach to design NNIs targeting disordered regions of NS5 has been proposed [ ].

The idea is to target the structural plasticity of NS5 to affect its activities. However, details on the disordered regions must be obtained to aim specifically and efficiently affect the protein activity. A challenging alternative for the identification of new specific compounds is to target the interaction network within the replication complex and with the host cell protein partners, with a view to disrupt key protein—protein interactions [ , , ].

An obvious target for such a strategy is to target the regions of interaction between the elements of the replication complex NS3-NS5, NS3-NS4 for example.

Assays to disrupt the interaction between NS3 and NS5 proteins are being developed [ ]. Another interesting approach would be to disrupt the interaction between with identified partners like Daxx or STAT2. This type of molecules would be very interesting if used in complement of an activity inhibitor of NS5 NIs or NNIs to enhance their potential. However, to screen for such compounds, robust assays need to be developed. The intrinsically disordered regions might in this case play an important role [ ].

This interaction enhances the viral replication. This example where expression of the host protein is aimed to affect virus replication is a good illustration of an antiviral strategy that would target critical host partners of viral proteins rather than the virus directly. Identifying the exact partners and characterizing their interactions with the replication would shed light on new antiviral targets to treat dengue infections.

National Center for Biotechnology Information , U. Journal List Viruses v. Published online Apr Author information Article notes Copyright and License information Disclaimer.

Received Jan 31; Accepted Apr This article has been cited by other articles in PMC. Abstract The World Health Organization estimates that the yearly number of dengue cases averages million. Keywords: flavivirus, dengue virus, NS5 polymerase. Introduction Dengue virus is a mosquito-borne human pathogen affecting mostly inter-tropical regions, where 3.

Open in a separate window. Figure 1. Figure 2. Methyltransferase Activity The methyl group donor is an S-adenosylmethionine molecule bound inside a deep pocket within the MTase domain. Figure 3. Linker Domain and Protein Flexibility Detailed information on the cooperation between NS5 and NS3 proteins and how the helicase and RdRp activities and the triphosphatase and capping activities are subjected to a cross-talk between the various domains is still lacking.

Figure 4. Host Interacting Partners In addition to its central role in genome replication, NS5 proteins from flaviviruses play a crucial part in evading the host immune system. Table 1 Examples of NS5 inhibitors.

Conflicts of Interest All the authors have declared no competing interest. References 1. Chatel-Chaix L. Cell Host Microbe. Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells. Rastogi M. Flavivirus NS1: A multifaceted enigmatic viral protein. Somnuke P. N -linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement.

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Upadhyay A. That zinc atom was proposed to contribute to structural stability of the region as well as regulating thumb subdomain conformational changes that occur during the reaction pathway [ 30 ]. A recent structural study also showed that the thumb subdomain was more dynamic than the finger and palm subdomains with greater deuterium-exchange as well as higher crystallographic temperature factors [ 28 ].

The authors also suggested that cross talk between the N-terminus domain and the fingers subdomain contributed to various conformational ensembles for interaction with various viral proteins, RNA and host cofactors during the virus replication cycle [ 28 ].

Therefore our identified cysteine modifications would impact structurally on several aspects of NS5 and these effects can be observed in changes for both guanylyltransferase activity and the RdRp activity. The plots were generated by GraphPad Prism version 5.

See S4 Fig for an example of the primary plot. Several reports suggest that virus induced oxidative stress may regulate host response through a protein post translational modification such as glutathionylation [ 29 , 33 ]. For dengue NS5 specifically it has been reported that different extents of phosphorylation control cellular localization in the cytosol or nucleus, as well as the ability of NS5 to interact with NS3 and function in viral RNA replication [ 34 , 35 ].

Recently dengue NS5 has also been reported to be modified by small ubiquitin-like modifier SUMO , a second reversible posttranslational modification that also regulates a variety of cellular systems [ 36 ]. As previously stated, protein glutathionylation is a reversible posttranslational modification that also has been reported to modulate many cellular processes.

In this report we have shown NS5 to be glutathionylated both for dengue as well as a second flavivirus Zika. The cysteine residues we found to be glutathionylated are highly conserved across multiple flaviviruses Fig 5. This suggests that glutathionylation may be a general feature for flavivirus NS5. We also show that this modification occurs multiple times on the protein and can modulation NS5 enzyme activities. How these modifications affect the various NS5 functions in the cell remains to be addressed.

After this period cells were washed 3 times with PBS to remove unabsorbed viruses. Fresh culture medium was added to the cells and cells were further incubated under standard conditions for 1 day. The virus was then removed and the cells were further incubated under standard conditions and harvested at 0, 2 and 6 h post infection hpi.

Cells were lysed and measured for protein concentration by Bradford method using BSA as standard. Cell pellets were incubated on ice and vigorously vortexed every 10 min for 30 min total time. The cell lysate supernatants were measured for protein concentration as described above. Anti-dengue virus prM glycoprotein antibody [DM1] from Abcam ab Anti-Dengue Virus 2 antibody from Abcam ab against Capsid protein. Mouse anti-glutathionylation antibody was from USBiological G The precipitates were collected by centrifugation at g for 5 min and the supernatants were discarded.

The IP was performed in 2 different ways. The first method was to incubate cell lysates with an anti-glutathione GSH primary antibody to pull down all glutathionylated proteins in the cell lysate and then probed with each anti-dengue virus antibody by western blot. The second method was for reverse confirmation by immunoprecipitating the dengue virus protein with the specific primary antibody and then probing with an anti-GSH antibody.

Proteins were transferred onto nitrocellulose membranes. The protein samples were run under non-reducing gel conditions no DTT if the anti-GSH primary antibody was used for analysis. The signals were detected with enhanced chemiluminescent detection reagents ECL. The virus was then removed and the cells were further incubated under standard conditions. Mock infected cells were generated in parallel.

The cell lysate supernatants were prepared and measured for protein concentration as described above. A non-treated sample was used as control. The first-strand cDNA was synthesized from 0. The forward and reverse primers of both NS5 genes were designed to contain Xho I and kpn I restriction sites. The recombinant plasmids were transformed into E. The candidate clones were first screened by a rapid size screening method before final verification by DNA sequencing.

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Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns.