Biochemical Analysis: Dengue Denv Protease Dengue Virus Protein Biochemical Analysis

Database Search Methods

Of the several CSU databases available, I chose to utilize PubMed, because Medline is specific for biological research. I also wanted to avoid retrieving qualitative studies, given the topic chosen, and felt Pubmed would be the best way to find quantitative studies.

The first search string I used was , which retrieves 4,028 citations. Since I was unfamiliar with this topic, I clicked on the "Review" filter at the top left of the window. After scanning through the titles I chose one and read the abstract (Morrison, Aguirre, and Fernandez-Sesma, 2012). The abstract for this article provided enough information about the protein complex DENV protease that I could begin to narrow my search. The next search string utilized was , which returned 56 citations. Since the instructions required articles published within the last five years, I began reading the most recent citation titles and found two that characterized the activity of the dengue NS2B-NS3 protease in the presence of putative protease inhibitors. I then investigated whether the search term <

S2B-NS3> would provide more interesting results. The results of this search revealed that a number of viruses produce this protease and therefore these search results were rejected. I then searched PubMed using the term and too few citations were retrieved. I returned to using the search term and then limited the retrieval to free full-text articles by clicking on the link in the upper right-hand corner of the window. I was able to discover two more citations investigating the mechanisms of DENV protease activity without any reference to putative protease inhibitor research in the abstracts.

3. Given the small number of citations retrieved using the search strategy outlined above, I did not need to use more complicated search strings like or "NS2B-NS3"[title] AND "Dengue."

4. Citations selected in APA citation style:

a. Rothan, Hussin A., Han, Heh Choon, Rmasamy, Thamil Selvee, Othman, Shatrah, Rahman, Noorsaadah Abd, and Yusof, Rohana. (2012a). Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1. BMC Infectious Diseases, 12, 1-9.

b. Rothan, Hussin A., Abdulrahman, Ammar Y., Sasikumer, Pottayil G., Othman, Shatrah, Rahman, Noorsaadah Abd, and Yusof, Rohana. (2012b). Protegrin-1 inhibits dengue NS2B-NS3 serine protease and viral replication in MK2 cells. Journal of Biomedicine and Biotechnology, 2012, 1-6.

c. Morrison, Juliet, Aguirre, Sebastian, and Fernandez-Sesma, Ana. (2012). Innate immunity evasion by dengue virus. Viruses, 4, 397-413.

5. Since I limited my search to free full-text articles available through PubMed Central, the above citations can be read online or downloaded as a PDF file.

Part B

Introduction

The more virulent viruses are typically able to evade a limiting innate immune response through some mechanism. The mechanism used by the dengue virus depends on the activity of the DENV protease complex, which has been shown to interfere with type I interferon production by the innate immune system (reviewed by Morrison, Aguirre, and Fernandez-Sesma, 2012). Since this is one of the first and most important signals recruiting other immune cells to the site of infection, the subsequent cascade of immune responses is subverted. Interfering with the formation of this complex, which consists of the protease protein NS3 and its cofactor NS2B, inhibits proteolytic cleavage of the viral RNA-derived polyprotein required for viral replication. Based on the results of computer modeling experiments, theta-defensins appear to be potential candidates for disrupting the interaction between NS3 and NS2B (reviewed by Rothan et al., 2012a). To test this theory, Rothan and colleagues (2012a) examined the protease activity of recombinant NS3 and NS2B in the presence of the primate defensin retrocyclin-1 (RC-1).

Experimental Method: Protease Assay

The NS3 protease has trypsin-like serine protease activity and requires association with the cofactor NS2B to have full activity (reviewed by Rothan et al., 2012a). Recombinant versions of both proteins and of the defensin RC-1 were produced in E....

coli and then assayed for protease activity using a fluorescent spectrophotometer plate reader. The substrate for this assay was Boc-Gly-Arg-Arg-AMC (Boc-GRR-AMC). The Boc (t-Butyloxycarbonyl) group stabilizes the substrate and the covalent bond with the peptide quenches the fluorescent of the AMC (amino methyl coumarin) molecule (Novabiochem, 2005). When exposed to a protease, the AMC fluorophore is freed from the peptide group and fluorescence increases significantly when excited at the appropriate wavelength.
In the study being reviewed here, protease activity was measured by an increase in fluorescence activity at 440 nm after excitation at 350 nm using a fluorescent spectrophotometer plate reader (Rothan et al., 2012a). The assay design chosen by Rothan and colleagues (2012a) is called a homogenous assay because the product or substrate does not need to be isolated from the reaction mixture before quantification (Zhang, 2012). The 30 minute incubation period prior to reading fluorescent activity is also consistent with enzyme dynamics that reach equilibrium rapidly.

The main ingredient for the protease essay was a single-chain recombinant protein containing the sequence of both NS3 and NS2B (NS2B-NS3pro) (Rothan et al., 2012a). By producing a single chain polypeptide, the authors avoided the need to perform an association step to bring NS3 and NS2B together. The other main ingredients were recombinant RC-1 and the substrate Boc-GRR-AMC. A Tris-HCl buffer was prepared (pH 8.5) that acted as the reference condition, because it should have minimal or undetectable fluorescent activity at 440 nm even in the presence of substrate. The second condition was buffer plus 2 ?M NS2B-NS3pro, which should produce fluorescence activity in the presence of substrate in a concentration-dependent activity. The results of these two conditions when incubated for 30 minutes at three different temperatures are shown in Figure 1. The three temperatures represent the conditions under which DENV protease will be expected to help the virus replicate, which are 28?C for a mosquito host, 37?C for an uninfected human host, and 40?C for an infected human host with a high fever. If fluorescence is produced in a concentration-dependent manner, then this is proof the assay works as expected.

The third condition was buffer with NS2B-NS3pro (2 ?M) and substrate (20 ?M) at constant concentrations, while titrating recombinant RC-1 (Rothan et al., 2012a). This assay was also performed at the three most relevant temperatures of 28?C, 37?C, and 40?C. If the fluorescent signal generated by NS2B-NS3pro cleavage of the substrate is inhibited in a concentration-dependent manner by RC-1, then this supports the theory that the defensin RC-1 can inhibit DENV protease in vivo and by extension, dengue viral replication.

The basic approach for conducting these assays would be to allocate a fixed volume to wells within a multi-well plate that can be read in the spectrophotometer (Rothan et al., 2012a). For the first experiment, there were three temperatures and six different concentrations of substrate. Since each experiment was run in triplicate, this would require 54 wells. The second experiment was performed at three different temperatures, in triplicate, with six different concentrations of RC-1 (null condition now shown in figure 4) and would therefore need 54 wells as well. The way this protease assay was designed enabled Rothan and colleagues (2012a) to determine the substrate Km values and the RC-1 IC50 (half maximal inhibition) at all three temperatures.

Results

Figure 3 shows the enzyme kinetics of NS2B-NS3pro acting upon the Boc-GRR-AMC substrate at the three temperatures (Rothan et al., 2012a). Six different NS2B-NS3pro concentrations were used, including buffer alone, which generated three temperature-dependent saturation curves. The highest protease activity was exhibited at the temperature of mosquitoes. The next highest activity was at normal human body temperature. Much lower activity was observed at 40?C, the body temperature of an infected individual running a high fever. The results shown in Figure 3 reveal the assay works as expected and that the DENV protease would be most active in mosquitoes. The NS2B-NS3pro Vmax is therefore temperature dependent.

Figure 4 shows the results of the RC-1 titration at the three…