Dengue Virus NS2B-NS3 Protease Biochemical Analysis

Database Search Methods

Of the several databases available, I chose to utilize PubMed, because MEDLINE is specific to 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 Dengue protein, which retrieved 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 used was DENV protease, which returned 56 citations. Since the assignment 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 NS2B-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 Dengue NS2B-NS3, but too few citations were retrieved. I returned to using the search term DENV protease 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.

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

Citations selected, in APA citation style:

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.

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.

Introduction to DENV Protease and Immune Evasion

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

Since the search was limited to free full-text articles available through PubMed Central, the above citations can be read online or downloaded as PDF files.

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 type I interferon signaling 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 Design

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 fluorescence of the AMC (aminomethyl 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 reviewed here, protease activity was measured by an increase in fluorescence 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 homogeneous 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 assay 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 a separate 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) to serve as the reference condition, because it should produce minimal or undetectable fluorescent activity at 440 nm even in the presence of substrate. The second condition was buffer plus 2 µM NS2B-NS3pro, which should produce fluorescence activity in the presence of substrate in a concentration-dependent manner. 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 would be expected to support viral replication: 28°C for a mosquito host, 37°C for an uninfected human host, and 40°C for an infected human host running a high fever. Fluorescence produced in a concentration-dependent manner serves as proof that the assay works as expected.