# Patent

## Title
Active truncated form of the RNA polymerase of flavivirus

## Abstract

The isolation and purification of two domains from a from a flavivirus is provided. Each domain can function independently. Moreover, one domain codes for a sequence that provide polymerase activity. A process for screening possible modulators of the polymerase activity of an isolated and purified polypeptide from flavivirus is also disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/474,670, filed May 30, 2003, the subject matter of which is fully incorporated herein by reference.


FIELD OF INVENTION
This invention relates to antiviral molecular biology. More particularly, it relates to the isolation and identification of an active truncated form of the RNA polymerase of flavivirus, capable of being easily reproduced, and serving as a target for high-throughput screening of antiviral drugs.
BACKGROUND
The genus flavivirus contains approximately 70 positive single-stranded RNA viruses, among which many major human pathogens are found, including Dengue virus (“DV”), West Nile (“WNV”), Yellow Fever virus (“YFV”), Japanese and tick-borne encephalitis viruses. YFV was the first flavivirus to be isolated in 1927, but historically, flavivirus-like diseases have been reported in the medical literature since at least 1780.
On of the most common and virulent flaviviruses is DV. DV threatens up to 2.5 billion people in 100 endemic countries. Up to 50 million infections occur annually with 500 000 cases of dengue haemorrhagic fever and 22,000 deaths mainly among children. Dengue has been classified by the World Health Organization (“WHO”) as a priority as it ranks as the most important mosquito-borne viral disease in the world. In the last 50 years, its incidence has increased 30-fold. Prior to 1970, only 9 countries had experienced cases of dengue haemorrhagic fever (“DHF”); since then the number has increased more than 4-fold and continues to rise.
WNV also has become much more wide-spread. In 1999, WNV was isolated for the first time in the Americas during an outbreak in New York City. By the end of 2002, WNV activity had been identified in 44 states of the United States and the District of Columbia. The 2002 WNV epidemic resulted in 4,156 reported human cases of WN disease including 2,942 meningoencephalitis cases and 284 deaths.
There have been previous attempts to generate a vaccine. For example, a live, attenuated virus of YFV (strain 17D) was developed in 1936 and has been used as a vaccine for over 400 million people. Unfortunately, the vaccine has not proved 100% successful since there are 200,000 estimated cases of yellow fever (with 30,000 deaths) per year worldwide, 90% of which in Africa. Chimeric live vaccines incorporating genes of either Japanese encephalitis, WNV, or Dengue in a YFV 17D vector are currently in development. However, a number of difficulties are associated with the conception of safe and efficient vaccines, such as vaccine purity, and immunogenic cross responses. That is why antiviral chemotherapy has a major role to play in the control of such diseases.
Since viral RNA polymerase is critical for replication of the virus and cannot be substituted by any other cellular polymerase, it is an excellent antiviral target. As a result, most of the more than 30 new antiviral agents, which have been developed and approved during the last 5 years, are directed against viral polymerases. They are mainly targeted against human immuno-deficiency virus, but drugs against hepatitis B and C, herpes simplex, varicella-zoster and influenza virus infections have also been made commercially available.
More than 50% of these antiviral agents are nucleoside analogues, in which the base, the ribose moiety or both have been modified. Nucleoside analogues can act as inhibitory ligands by binding to the template binding site within the polymerase active site and preventing the access of the viral RNA, or by binding to the nucleotide binding site, thus limiting the availability of the natural substrate for complementary strand synthesis. It is generally understood in the art that a nucleoside analogue may be a synthetic molecule that resembles a naturally occurring nucleoside, but lacks a bond site needed to link it to an adjacent nucleotide. Additionally, nucleoside analogues can also act as chain-terminators during DNA or RNA synthesis, by binding themselves as a substrate for the target polymerase, but preventing further chain elongation. Non-nucleoside analogues may bind to allosteric sites thus influencing the local conformation of the active site via long-range conformational changes of the polymerase's structure.
Another approach whereby many antiviral compounds have been discovered is by using cell cultures infected with the virus of interest. In such cases, addition of an antiviral compound protects the cells from infection, or inhibits virus growth. For this type of experiment, it is useful to identify a large number of antiviral compounds in an efficient manner. As such, another evolving mechanism to identify new antiviral agents through the high-throughput screening (“HTS”) of a large number of synthetic or natural compounds. This requires the development of an in vitro assay, which in turn requires large amounts of soluble and active protein.
When a high number of potentially antiviral compounds are tested by HTS, it is possible to identify antiviral compounds in an efficient manner. This approach has been used successfully for HIV and other viruses. However, in some cases, this approach is difficult due to the absence of a suitable system allowing infection of a cell in vitro.
In other cases, even if a suitable cell-based assay is available, this procedure may be too cumbersome or expensive. This is the case for certain dangerous viruses—such as those that require BSL-3 and/or BSL-4 facilities. Establishing a screening process for over a large amount of compounds in a BSL-3 or BSL-4 containment facility has not been achieved yet because of this heavy expense and burden. For example, flaviviruses belong to this class of viruses. These viruses require from BSL-2 to BSL-4 facilities (e.g., Dengue, WNV and/or Kyasanur Forest viruses). Thus, in such cases, it is preferable to screen potentially antiviral compounds directly on viral target proteins.
For efficiency, especially considering the difficulty with certain, more dangerous viruses, the characterization in molecular terms of the target, the viral polymerase, is of prime importance in the screening and selection of antiviral compounds. In the case of the flavivirus RNA polymerase (“NS5” or sometimes referred to herein as “NS5Pol”), this task has proven to be difficult for several reasons. First, polymerase genes have been notoriously difficult to clone in their entirety. When available, recombinant NS5 has been reported to be unstable in bacterial hosts. In addition, the notoriously low yield of soluble purified NS5 is a limiting factor to set up polymerase-activity assays. Another possible reason for the described difficulties is the fact that NS5 does not carry a single enzymatic activity.
Very recently, we described an N-terminal domain of NS5 (sometimes referred to herein as “NS5 methyltransferase domain”) which acts as an S-adenosyl-L-methionine (AdoMet)-utilizing RNA-cap 2′Omethyltransferase, thus participating in mRNA capping, which is generally understood as the process of adding a guanosine nucleotide to the 5′ end of mRNA (the methelyated end of guanosine) (Egloff & Benarroch, 2002). Additionally, we showed that the NS5 methyltransferase domain binds GTP analogues.
Due to the nature and proximity of the NS5 methyltransferase domain to the polymerase domain of the flavivirus, the description and characterization of the NS5 methyltransferase domain clearly shows that some nucleoside analogues and inhibitors of flavivirus replication could potentially be, in fact, mRNA-capping inhibitors without any effect on the polymerase activity. Likewise, it is very possible to mistakenly identify a compound as binding to NS5 and characterizing the binding data as potentially interesting for inhibition of the polymerase, but, in reality, only the RNA-capping has been affected. Therefore, it would be useful to identify and define the “junction” or sequence between the NS5 methyltransferase domain and the polymerase domain.
SUMMARY OF THE INVENTION
This invention relates to the isolation and purification of a polypeptide from a flavivirus.
In another aspect of the invention, the polypeptide can be separated into two domains, the N-terminal domain and the C-terminal domain, both of which are separately active.
In another aspect of the invention, the junction between the N-terminal and C-terminal domains has been identified.
In yet another aspect of the invention, the results indicated that independent expression of each of the separated domains provided greater expression than the full, unseparated polypeptide.
In still another aspect of the invention, the C-terminal of the domains in particular is purified and acts as active RNA polymerase.
In yet another aspect of the invention, the C-terminal domain demonstrates substantial homology with other RNA polymerases of clinical interest.
In still another aspect of this invention, the polymerase provides a surrogate model and system to screen synthetic and natural compounds against the polymerases of related viruses.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the sequence alignment of NS5 of flaviviruses: Dengue2 (SEQ ID NO. 1); Dengue3 (SEQ ID NO. 2); Dengue1 (SEQ ID NO. 3); Dengue4 (SEQ ID NO. 4); WestNile (SEQ ID NO. 5); Kunjin (SEQ ID NO. 6); JapaneseEncephalitis (SEQ ID NO. 7); YellowFever (SEQ ID NO. 8); Banzi (SEQ ID NO. 9); Langat (SEQ ID NO. 10); Powassan (SEQ ID NO. 11); Tick-borneEncephalitis (SEQ ID NO. 12); LoupingIll (SEQ ID NO. 13); Modoc (SEQ ID NO. 14); RioBravo (SEQ ID NO. 15).
FIG. 2 shows two Western blots illustrating the expression and purification of NS5DV and NS5PolDV.
FIG. 3 shows a Western blot (FIG. 3A) and a graph (FIG. 3B) of the purified NS5PolDV.
FIG. 4 shows two graphs (FIG. 4A and FIG. 4B) demonstrating the activity level of NS5PolDV on poly(rC).
FIG. 5 shows a steady-state Km determination of GTP with and poly(rC) (in FIG. 5A) and for monomeric NS5PolDV using poly(rC) without primer (de novo initiation) (in FIG. 5B).
FIG. 6 shows divalent cation optimum curves for NS5PolDV and NS5PolWNV (in FIG. 6A) on poly(rC) without primer (de novo initiation) (in FIG. 6B).
FIG. 7 shows two graphs demonstrating the activity level of of NS5PolDV on specific heteropolymeric RNA templates.



DETAILED DESCRIPTION
Definitions
“Structural equivalents” should be understood to mean a protein maintaining its conformational structure as if the protein were the native protein expressed in its natural cell.
“Substantial homology” or “substantially homologous” means a degree of homology between the isolated and described NS5Pol (as defined herein) and the RNA polymerases of other positive-single-stranded RNA viruses of clinical interest when there is homology at least about 65%, preferably at least about 70%, most preferably in excess of 80%, and even more preferably in excess of 90%, 95% or 99%.
Results and Findings
In one aspect of this invention, we discovered a way to circumvent the above-described problems associated with viral polymerase. We performed a structural analysis of flaviviruses NS5 genes using biocomputing methods, and isolated and defined two unique domains of NS5. As described in the literature, two distinct domains are generally defined for the large family of flavivirus NS5 genes, and related structural equivalents.
Specifically, as shown in the experiments, NS5 was separated into the two domains using genetic engineering techniques. We have established the independent folding of these two putative domains using various methods. Moreover, demonstrated in our experiments as set forth below, each domain is separately active, and an appropriate ligand may be mapped to either the N-terminal (capping) domain or the C-terminal (polymerase) domain of NS5. These genetic constructs allow the production of higher quantities of either domain compared to the full-length protein. Thus, simply put, the C-terminal polymerase domain of NS5 (NS5Pol) of DV (subtype 2, Strain New Guinea C), WNV (strain New York 99) and the Kunjin variant of WVN (KV) are easy to purify in large quantities, they are active as a polymerase, and constitute one aspect of our invention.
As noted, the availability of large quantities of NS5Pol allows its use as a target in HTS. One of the advantages of the isolation of the polymerase domain is that the antiviral compound, which demonstrates the modulating activity of the polymerase domain, is specific to the polymerase activity of the viral protein, without any interference of the other parts of the protein. Indeed, it is possible to detect RNA polymerase activity in a single tube using standard radioactive or nonradioactive methods.
As described herein, modulation of the polymerase activity of the protein is important in creating antiviral agents for the treatment of the enumerated viral diseases. Since DV, WNV and KV NS5Pol domains are significantly homologous to and demonstrate substantial homology with NS5Pol domains of other flaviviruses, and since DV, WNV and KV NS5Pol are functionally homologous to the RNA polymerases of other positive-single-stranded RNA viruses of clinical interest (such as the related NS5B polymerase of HCV), NS5Pol provides also a surrogate model and system to screen synthetic and natural compounds against such related viruses. Simply put, the invention includes a method of screening antiviral compounds able to modulate the polymerase activity of significantly and functionally homologus NS5 gene encoding viruses (i.e. flaviviruses).
Expression and Purification
Based on preceding structural and functional studies on a N-terminal methyltransferase or capping domain of protein NS5 of flavivirus (Egloff and Benarroch, 2002) we predicted the limit of a functional and soluble C-terminal polymerase domain of NS5. In particular, FIG. 1 identifies the sequence alignment of various flaviviruses as follows: Dengue 2 (P14340) (SEQ ID NO. 1), Dengue 3 (Q99D35) (SEQ ID NO. 2), Dengue 1 (Q8VBS3) (SEQ ID NO. 3), Dengue 4 (AAA42964) (SEQ ID NO. 4), West Nile virus (AAL87234) (SEQ ID NO. 5), Japanese encephalitis virus (Q82872) (SEQ ID NO. 7), Yellow fever virus (Q89277) (SEQ ID NO. 8), Banzi virus (Q67483) (SEQ ID NO. 9), Langat virus (Q9IG40) (SEQ ID NO.10), Tick-borne encephalitis virus (Q8VBS4) (SEQ ID NO. 12), Louping ill virus (O10383) (SEQ ID NO. 13), Modoc virus (CAC82912) (SEQ ID NO. 14) and Rio Bravo virus (Q9JAD5) (SEQ ID NO. 15) were aligned by ClustalW. The secondary-structure elements of the NS5MTaseDV structure as determined by X-ray crystallography and of NS5PolDV as predicted by PredictProtein are displayed in black and red, respectively, above the sequence of NS5 Dengue. NS5Pol starts after the vertical bar just before a predicted alpha-helix. The remaining ca. 550 residues of NS5 are not shown.
As set forth above, FIG. 1 shows the sequence alignment of the N-terminal part of NS5 of several flaviviruses with the secondary structure elements of the capping domain of Dengue NS5 given above. Essentially, as discussed, the junction between the methyltransferase and polymerase portion of the flaviviruses was isolated, which allows the precise and efficient separation of the domains. The junction (or where the Pol domain starts) is located at amino acid 272.
Proteins NS5 and their corresponding Pol domains of DV, KV and WNV were expressed as recombinant proteins bearing a His-tag which facilitates subsequent purification. Accordingly, they were purified with immobilized metal-affinity chromatography (IMAC) in a first purification step. As noted above, FIG. 2 illustrates the superiority of NS5PolDV over NS5DV in terms of purification yield after IMAC following the same protocol. The results shown in FIG. 2, the expression and purification of NS5DV and NS5PolDV, were obtained as follows: NS5DV and NS5PolDV were cloned in expression plasmid pQE30, expressed in BL21[pDNAy] overnight at 17° C. after induction with 50 mM IPTG, addition of 2% EtOH and a cold shock (30 min at 4° C.). Sonication was done in 50 mM sodium phosphate lysis buffer, pH 7.5, 300 mM NaCl, 10% glycerol (10 ml of this lysis buffer for 3.6 g cell pellet) in the presence of DNAse, PMSF, protease inhibitors and lysozyme. Recombinant proteins were bound to metal-affinity-chromatography resin talon (Clontech) and eluted with 500 mM imidazole. SDS-PAGE of protein samples from expression and purification: upper panel: NS5DV, lower panel: NS5PolDV, lane 1: total fraction after lysis, lane 2: soluble fraction after lysis and centrifugation, lane 3: flowthrough of metal-affinity column, lane M: molecular mass markers, lanes 4 to 8: eluted fractions from metal-affinity column, F1 to F5, lane 9: metalaffinity resin. The corresponding molecular masses in kD of the markers are given on the left. NS5DV results in low purification yields due to instability resulting in the presence of a protein band the size of which corresponds to the polymerase domain (see arrow in the upper panel).
The low yield of NS5 is attributed to lower solubility of the recombinant protein and an elevated sensitivity to proteolytic cleavage during purification. This is illustrated in FIG. 2 by the presence of a cleavage product of around 73 kD which could represent the Pol domain. Yields for both proteins are compared in Table 1.







TABLE 1







Yield after expression and purification










Yield (mg per liter expression culture)












protein
after IMAC
after heparin















NS5PolDV
2
1.2



NS5DV
0.2
n.d.



NS5PolKV
10
7



NS5KV
7
0.6



NS5PolWNV
12
8



NS5WNV
n.d.
n.d.










A second purification step consists of heparin affinity chromatography. The results of this purification were illustrated in FIG. 3 and obtained pursuant to the following procedure: NS5PolDV eluates from metal-affinity chromatography were dialyzed against 50 mM sodium phosphate buffer, pH 7.5, 150 mM NaCl, 10% glycerol and submitted to heparin-affinity chromatography applying a salt gradient of 150 mM to 1M NaCl. Pure protein was eluted in two peaks, at 390 mM and 460 mM NaCl.
A: SDS-PAGE of protein samples from purification steps, lane 1: pooled protein fractions from metal-affinity chromatography after dialysis, lane M: molecular mass markers, lane 2: peak 1 from heparin-affinity chromatography, lane 3: peak 2 from heparin-affinity chromatography, lane 4: flowthrough from heparin-column. The corresponding molecular masses in kD of the markers are given on the left.
B: Analytical gel filtration (Superdex 200, Pharmacia) of peak 1 and 2 of NS5PolDV from heparin affinity chromatography. The elution volume of peak 1 corresponds to the monomeric form of NS5PolDV whereas peak 2 elutes earlier corresponding to oligomeric NS5PolDV (trimer or tetramer).
For NS5PolDV it results in two fractions eluting at different salt concentrations both representing NS5PolDV, as shown in FIG. 3A. Analytical gel filtration showed that peak 1 from heparin affinity chromatography represents the monomeric form of NS5PolDV whereas peak 2 represents an oligomeric form as shown in FIG. 3B. Both forms are purified to 98% after heparin purification step, the combined yield of NS5PolDV is 1.2 mg starting from one liter expression culture (Table 1).
Expression and purification of NS5 KV and NS5PoIKV follow a similar tendency compared to Dengue NS5 (sequence identity of NS5 66.4%). Although full-length NS5 KV and NS5PolKV render considerably higher yields compared to the corresponding Dengue proteins, still, full-length NS5 KV shows lower yields after one purification step (Table 1) and, due its instability, dramatically lower yields after a second purification step. In difference to NS5PolDV, NS5PolKV elutes as a single peak after heparin affinity chromatography (data not shown). The same applies to NS5PolWNV (sequence identity to NS5PolKV 94.6%).
In all cases, the final purification product, for which the purity is adequate for HTS assays, is purified with a >10-fold increase in yield compared to the unengineered polymerase.
Activity Data
Polymerase activity on NS5Pol was measured on homo- and heteropolymeric templates.
Homopolymeric Template
Activity was tested on three homopolymeric templates: poly(rC), poly(rU) and poly(rA). Only poly(rC) resulted to be a productive template for NS5PolDV. This was illustrated in FIG. 4 based on the following protocol: RNA polymerase activity was tested on a homopolymeric RNA template (polycytidylic acid, Amersham Biosciences) of an average length of 360 nt. A standard assay was carried out in 50 mM HEPES buffer, pH 8.0, 10 mM KCl, 5 mM MgCl2, 5 mM MnCl2, 10 mM DTT containing 1 μM template, 4 mM primer GG, 10 μM GTP, 0.01 mCi [3H]-GTP per μl reaction mixture and the concentration of enzyme given below. Reactions were carried out at 30° C. for given time periods and stopped by spotting a sample on DEAE filter discs (Whatman) presoaked with 50 mM EDTA. Filters were washed 3×10 min with 300 mM (NH4)2SO4 buffer, pH 8.0 and 2×5 min with EtOH and air-dried. Liquid scintillation fluid was added and incorporation in counts per minute (cpm) determined by using a Wallac MicroBeta TriLux Liquid Scintillation Counter.
A: Influence of specific E. coli RNA polymerase inhibitor rifampicin on NS5PolDV and E. coli polymerase (control). NS5PolDV was tested using the conditions given above at 64-nM enzyme concentration. E. coli RNA polymerase was obtained from USBiochemicals and used in NS5PolDV standard rection buffer at 37 nM.
B: Time course of [3H]-GTP incorporation by NS5PolDV peak 1 (monomeric preparation) and peak 2 (oligomeric preparation) from heparin 11 affinity chromatography (see FIG. 3A) tested at 80 nM enzyme concentration using poly(rC) without primer.
FIG. 4A shows incorporation of radioactively labeled GTP into a nascent poly(rG) polymerization product by NS5PolDV (oligomeric preparation) using primer rGG. In a control reaction E. coli RNA polymerase-inhibitor rifampicin was added and did not show any inhibitory effect. Thus, the observed incorporation of radioactive GTP in a polymerization product is not due to a contamination with E. coli RNA polymerase activity. FIG. 4B shows a parallel test of the oligomeric and monomeric preparation of NS5PolDV. In this experiment no primer was used, thus, polymerization is initiated de novo. Both preparations show identical catalytic efficiency. There are two possible explanations, either the state of oligomerization does not influence polymerase activity or under the conditions of the activity test (80 nM enzyme 6 concentration) both preparations adopt the same oligomerization state. In either way, both preparations can likewise be used for inhibitor screening studies.
In FIG. 5, the steady-state Km determination of GTP with various ligands was performed as follows: RNA polymerase activity was tested under conditions given in the description set forth above in the experiments related to FIG. 4 without the use of a primer. Initial velocities were determined over a time period of 5 min. GTP concentrations in the range of 2 to 500 μM were tested using poly(rC) at 1 μM. Template poly(rC) was tested in the range of 2 to 1000 nM with GTP fixed at 200 μM.
A: Plot of apparent intitial velocity (viapp in cpm per min) against GTP concentration. Data were fitted to a Michaelis-Menten hyperbola (viapp=Vmax[S]/(Km+[S]) using Kaleidagraph.
B: Plot of apparent initial velocity against poly(rC) concentration. Data were fitted as in A.
The Km of GTP was determined for NS5PolDV (monomeric preparation) as being 27.3±5.1 mM (FIG. 5A). Km values are in a similar range for the oligomeric preparation of NS5PolDV (66.2±6.6 mM) and for full-length NS5DV (13.0±2.9 mM). Thus, the affinity of NS5PolDV from both preparations versus the substrate GTP that binds to a nucleotide-binding site within the active site of the enzyme are close. This again confirms that both preparations can be used likewise. Additionally, the fact that NS5PolDV shows similar substrate affinity as NS5 indicates that the polymerase domain of flavivirus NS5 is a valid model system for the identification of inhibitors of the full-length protein. The Km values of polyC were determined for the monomeric preparation of NS5PolDV as 17.3±2.4 mM, the oligomeric preparation as 14.8±3.8 mM and for NS5DV as 18.0±7.3 mM. As seen above, the corresponding plot for the monomeric preparation of NS5PolDV is shown in FIG. 5B. First, this allows the conclusion that a putatively different oligomerization status of NS5PolDV does not seem to influence the affinity to a long template which could be expected to maintain cooperative interactions with two enzyme molecules at the same time. Secondly, template poly(rC) (around 360 nt long) shows identical affinity to NS5DV and NS5PolDV within the context of its use as a polymerization template. Thus, the capping domain does not seem to be involved in template binding indicating again that the isolated polymerase domain can be used for inhibitor screening experiments of the polymerase activity of full-length NS5.
FIG. 6A shows the optimum curve for divalent manganese (Mn) and magnesium (Mg) ions of NS5PolDV on poly(rC). Polymerization on poly(rC) works exclusively in the presence of Mn ions. This is also the case for NS5PolWNV as shown in FIG. 6B. The results set forth in FIG. 6 were obtained pursuant to the following protocol:
A: Tests of NS5PolDV were carried out under standard conditions given in the description set forth above in the experiments related to FIG. 4 at 60 nM enzyme concentration. Influence of Mn2+ was tested either in absence of Mg2+ (Mn) or in presence of 5 mM Mg2+ (Mn (5 mM Mg)). Mg2+ was tested in the absence of Mn2+ (Mg).
B: Test of NS5PolWNV were carried out under the same reaction conditions as for NS5PolDV with the exception of GTP which was used at 100 μM. NS5PolWNV concentration was 400 nM.
Clearly, as seen from FIGS. 6A and 6B, the optimum curves for Mn2+ show that NS5PolWNV tolerates higher Mn2+ concentrations for the polymerization on polyC than NS5PolDV. The apparent Km value for substrate GTP is around 2 to 4-fold higher (130 mM) for NS5PolWNV and NS5PolKV in comparison to NS5PolDV (data not shown). These mechanistic differences between flavivirus polymerases can be studied in detail using well expressed and stable independent NS5Pol domains.
Heteropolymeric Template
NS5PolDV activity was tested on heteropolymeric specific templates comprising 717 nucleotides (225 nt of the 5′ and 492 nt of the 3′ of the Dengue genome). Specifically, the results are demonstrated in FIG. 7 and were obtained pursuant to the following protocol: Heteropolymeric RNA templates of 717 nt were generated by in vitro transcription using T7 RNA polymerase. The DNA template containing 225 nt of the 5′ and 492 nt of the 3′ of Dengue genomic RNA was constructed into plasmid pUC18. PCR products with the T7 promoter on the 5′ of the positive-sense strand or the 5′ of negative-sense strand were generated and used as substrates for in vitro transcription. RNA templates were replicated by NS5PolDV in 50 mM HEPES buffer, pH 8.0 containing 10 mM KCl, 10 mM DTT, 100 nM RNA template, 200 nM NS5PolDV, 500 mM ATP, UTP, GTP, 10 mM CTP and [a-32P]-CTP at 0.1 mCi/ml. Reactions were carried out at 30° C. and stopped by spotting a sample on DEAE 12 filter discs (Whatman) pre-soaked with 50 mM EDTA. Filters were treated as explained above in the experiments related to FIG. 4.
A: Comparison of incorporation on positive-sense and complementary negative-sense minigenome RNA templates. Reactions were carried out as given above except for the use of 500 mM CTP, 50 mM GTP, 0.1 mCi/ml [a-32P]-GTP and 5 mM Mn2+.
B: Divalent-cation optimum curves on positive-sense specific RNA template. Mn2+ was used in the absence of Mg2+ and, likewise, Mg2+ in the absence of Mn2+. Reaction were stopped after 60 min. Incorporation of CMP is given in cpm. The axis on the left corresponds to values obtained in presence of Mn2+ and the axis on the right to values obtained in presence of Mg2+.
This “minigenome” template illustrated in FIG. 7 contains secondary-structure and sequence elements necessary for efficient 7 de novo initiation and replication. Positive-sense and negative-sense RNA was generated by in vitro transcription from PCR products containing the promoter for T7 RNA polymerase in either sense. Specifically, FIG. 7A illustrates that NS5PolDV replicates negative-sense mini-genome RNA with higher efficiency compared to positive-sense RNA. This observation is in accordance with the observation that 10-times more positive-sense genomic RNA is produced in Dengueinfected cells in comparison to negative-sense RNA. Optimum divalent cation concentration (Mn2+ and Mg2+) was determined for replication of the positive-sense template (FIG. 7A). NS5PolDV replicates the template with 20-fold higher efficiency at optimal Mn2+ concentration compared to optimal concentration of Mg2+. The affinity of Mn2+ to active site residues seems to be higher compared to Mg2+ as the optimum concentration is lower.
Selective Inhibition of Dengue Virus Polymerase Activity Using Nucleotide Inhibitors
GTP analogs were used on NS5PolDV and NS5PolWNV to demonstrate their capacity to inhibit the NS5 polymerase domain using the homopolymeric template poly(rC). IC50 values were determined using GTP concentrations close to the determinded Km values (10 mM for NS5PolDV and 100 mM for NS5PolWNV). Table 2 shows the determined values of three putative chain terminators (3′-deoxy GTP, 3′-dioxolane 3′-deoxy GTP and 2′,3′-dideoxy GTP) and 2′-O-methyl-GTP which is expected to be incorporated into the growing RNA chain, thus acting as a competitive inhibitor.







TABLE 2







IC50 values for selected GTP analogs


Reactions on poly(rC) were done under conditions given in FIG. 4


without a primer. NS5PolDV was used at 60 nM. Incorporation of


[3H]-GTP was measured after 15 min. Inhibitors (TriLink Corp.)


were tested in the concentration range of 1 nM to 100 mM.










IC50 (μM)












inhibitor
NS5PolDV
NS5PolWNV















3′-deoxy GTP
0.02
0.18



3′-dioxolane 3′-deoxy GTP
1.2
58



2′,3′-dideoxy GTP
0.8
30



2′-O-methyl-GTP
5.6
105










The capacity of 2′-O-methyl-GTP (10 mM) was compared to inhibit NS5PolDV (dimeric preparation) and full-length NS5DV. Initial velocities were determined on poly(rC) at 10 mM GTP and compared to corresponding values without inhibitor. Initial velocities were determined to be 29.3% for NS5PolDV (dimeric preparation) and 19.3% for NS5DV compared to the proteins without inhibitor. This indicates that the NS5PolDV preparation is less inhibited than the full-length polymerase. Since these inhibition results are not identical, questions related to the putative interference of the capping domain remain when using the full-length polymerase.
It is clear that the removal of the capping domain (methyltransferase domain) which is able to bind GTP and GTP analogs, at the N-terminus of NS5 provides the opportunity to obtain unambiguous data which will show that the present truncated polymerase is the target of these inhibitors. The same applies to non-nucleoside inhibitors.
CONCLUSION
To summarize our results: the polymerase domain of flavivirus NS5
1. is purified with higher yields than full-length NS5. The overall increase in yield of the purified product suitable for HTS assays is >10-fold, facilitating mass production for HTS assays.
2. is much more stable than full-length NS5, as less protein is lost or degraded during the course of purification, giving a cleaner and more homogeneous reagent.
3. is active on homopolymeric and heteropolymeric specific templates. Thus, the capping domain is not necessary for the polymerase activity defined as the capacity to initiate and incorporate nucleotides into RNA. The polymerase domain defined here is a bonafide polymerase useful to conduct drug-screening assays.
4. shows similar affinities for template and substrates as the full-length NS5, and is identical in all points tested in terms of polymerase activity.
5. is unambiguous about being the target of a potential inhibitor. As the capping domain is not present, inhibition of NS5 function by GTP analogues and other molecules cannot be accounted for by inhibition of the capping domain or indirect inhibition of the NS5 polymerase activity by interference of the inhibitor with the capping domain.
6. is a valid polymerase model to conduct inhibitor screening studies with the aim to identify putative inhibitors of flavivirus RNA polymerases.

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## Claims

1. An isolated and purified polypeptide from a flavivirus having polymerase activity, wherein the full-length amino acid sequence of said polypeptide has at least 80% of identity with the SEQ ID NO: 16.

2. The isolated and purified polypeptide of claim 1, wherein the full-length amino acid sequence of said polypeptide has at least 95% of identity with SEQ ID NO:16.

3. An isolated and purified polypeptide from a flavivirus having polymerase activity, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

4. An isolated and purified polypeptide comprising the polymerase domain of the C-terminus of the NS5 polypeptide of a flavivirus, wherein said polypeptide has having polymerase activity and wherein the full-length amino acid sequence of said polypeptide has at least 80% of identity with the SEQ ID NO: 16.

5. The isolated and purified polypeptide of claim 4, wherein said polypeptide is selected from the group consisting of the polymerase domain of the C-terminus of the NS5 polypeptide of the Dengue 1, Dengue 2 and Dengue 3.