Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-06-20
2002-10-08
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S440000
Reexamination Certificate
active
06461845
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates Hepatitis-C virus (HCV), specifically to expression and purification of an RNA-dependent RNA polymerase (RDRP) encoded by the HCV genome, to antibodies directed against HCV-RDRP and to methods of using the enzyme to diagnose chronic HCV infections and to screen for antiviral agents effective against HCV.
BACKGROUND OF THE INVENTION
HCV is the major causative agent for post-transfusion and for sporadic non A, non B hepatitis (Alter, H. J. (1990)
J. Gastro. Hepatol
. 1:78-94; Dienstag, J. L. (1983)
Gastro
85:439-462). Despite improved screening, HCV still accounts for at least 25 % of the acute viral hepatitis in many countries (Alter, H. J. (1990) supra; Dienstag, J. L. (1983) supra; Alter, M. J. et al. (1990a)
J.A.M.A
. 264:2231-2235; Alter, M. J. et al (1992)
N. Engl J. Med
. 327:1899-1905; Alter, M. J. et al .(1990b)
N. Engl J. Med
. 321:1494-1500). Infection by HCV is insidious in a high proportion of chronically infected (and infectious) carriers who may not experience clinical symptoms for many years. The high rate of progression of acute infection to chronic infection (70-100%) and liver disease (>50%), its world-wide distribution and lack of a vaccine make HCV a significant cause of morbidity and mortality.
HCV is an enveloped virus whose genome is a 9.5 kb single-stranded RNA (sense(+)) encoding a single polyprotein that is processed by proteolysis to yield at least 9 proteins. HCV is related to pestiviruses and flaviviruses (Choo, Q-L. et al. (1989)
Science
244:362-364; Choo, Q-L. et al. (1991)
Proc. Natl. Acad. Sci. USA
88:2451-2455. Reinfection of previously HCV-infected chimpanzees suggests that protective immunity is transient or non-existent (Farci, P. et al (1992)
Science
258:135-140). Furthermore, results of recent vaccine trials suggest that development of an effective vaccine is remote (Houghton, M. et al. (1994) 2nd Internat. Meeting on Hepatitis C (San Diego)). Attempted treatment of chronic HCV infection using existing antiviral agents produces low cure rates and serious side effects. (Dienstag, J. L. (1983) supra.)
The nucleotide sequence of the HCV genome has been cloned and a single open reading frame has been identified. Using a vaccinia virus expression system, several cleavage products have been tentatively identified. (Lin, C. et al. (1994)
J. Virol
. 68:5063-5073; Grakoui, A. et al. (1993)
J. Virol
. 67:1385-1395.) The various putative cleavage products were recognized by antibodies raised against various peptides synthesized from amino acid sequences deduced from various segments of the coding regions. Sizes of antibody-reactive peptides were estimated by SDS-PAGE (See FIG.
1
). The non-structural protein designated 5B (NS5B) has been shown to have an amino-terminal sequence SMSY (Ser-Met-Ser-Tyr). The NS5B region encodes a 68 kd protein (p68) which contains an internal GDD (Gly-Asp-Asp) motif found in RNA-dependent RNA polymerases of other RNA viruses (Koonin, E. V. (1991)
J. Gen. Virol
. 72:2197-2206). However, no polymerase activity has been detected for HCV p68. In fact, the question has been raised that the 5B protein (p68) alone does not encode an active RNA-dependent RNA polymerase enzyme and that another subunit, possibly the NS5A gene product, is essential to catalytic activity. Prior attempts by the inventors and others to express the NS5B coding region as a fusion protein, using existing expression systems that facilitate purification of the fusion product and specific cleavage have failed to yield any active polymerase.
HCV, in common with other RNA viruses that employ direct RNA-RNA replication, has a high mutation rate. Independent isolates of HCV RNA have numerous sequence differences. Hagedorn, et al., (2000)
Curr. Top. Microbiol. Immunol
. 242:225-260, reviewed sequence variation in the NS5B sequence of 48 independent isolates. While it was possible to identify regions of conserved sequence, the interpretation of the data is difficult because only a few were known to encode an active RDRP. Even fewer were known to be a sequence of an infectious virus.
At the present time, infectivity of a given HCV strain can only be demonstrated in tests in chimpanzees, which severely limits the number of strains which can be tested. The number of RDRP sequences which have been tested for activity is limited, as described herein, by the necessity of modifying the N-terminus of the NS5B sequence to permit independent expression of RDRP in a recombinant host cell. Subsequent to the original priority hereof, others have isolated and expressed active RDRP. Lohmann, V., et al (1997)
J. Virol
. 71:8416-8428 GenBank Z97730, reported an active clone of HCV type 1b. The enzyme was shown to be active after deletion of either 25 or 55 amino acids of the C-terminus. Addition of an oligo-his tag permitted purification by nickel affinity chromatography of enzyme expressed in insect cells. Yamashita, T., et al (1998)
J. Biol. Chem
. 273:15479-15486 prepared an active RDRP from a clone of type 1b-JK1 (GenBank X65196). The authors thereof prepared a C-terminally deleted (&Dgr;21) RDRP fusion protein with glutathione-S transferase (GST) attached at the N-terminus. The enzyme was expressed in
E. coli
, yielding active enzyme with or without the GST tag, which served as an affinity purification ligand. The authors also reported three single amino acid replacements which abolished activity. Ferrari, E., et al (1999) reported several C-terminal deletion constructs &Dgr;16,19 21, 55 and 63 combined with oligo-his tags at either the N- or C-terminus. The enzymes were expressed in
E. coli
cells and purified with a chelated nickel column. RDRP enzymes of two HCV strains were studied, a 1a “H77” or “Hutchinson” isolate and a 1
b
“BK” isolate, although the RN sequences of the strains were not specifically identified. The oligo-his tags did not destroy activity, however enzymes having a C-terminal oligo-his tag had greater activity in an assay using a poly(C) homopolymer template. Luo, G.et al,(2000)
J. Virol
. 74:851-863 reported isolation of several NS5B clones from serum of an infected patient. Nearly half of the isolates had little or no RDRP activity. One isolate, which had the highest in vitro activity, was found to have a stop codon resulting in a deletion of 18 C-terminal amino acids. Mn
++
was found to stimulate activity 20-fold compared to the activity in the presence of Mg
++
. No nucleotide or amino acid sequences were reported. Patent publication No. WO 99/29843 disclosed an isolated NS5B sequence and encoded RDRP, both full length and having a 21 amino acid deletion at the C-terminus. The source of the HCV was not given although the sequences appear to be related to type 1a. No data regarding activity of the encoded RDRP was disclosed. For a recent review, see Hagedorn, C. et al. (2000).
SUMMARY OF THE INVENTION
The present invention provides methods for making modified structures of the HCV-RDRP. The need to make modifications is due to the quasi-species nature of the virus, the fact that the protein appears intracellularly as the product of post-translational cleavage of a polyprotein, and in vitro insolubility of the isolated enzyme. The modifications described herein enable translation of the NS5B region of HCV RNA in transformed host cells, without the necessity of translating other virus-coded proteins at the same time. The second category relates to modifications at the C-terminus that contribute to solubility of the enzyme in aqueous media. The third category relates to individual amino acid substitutions which can be introduced to individual isolates encoding HCV-RDRP to enhance enzyme properties. As a consequence of the high mutation rate that occurs during HCV replication, individual isolates encode RDRP variants that vary in primary sequence and in functional attributes. These include, for example, reaction rate, template specificity, processivity, ease of purification, stability during purification and during storage and the like. Other advant
Emory University
Greenlee Winner and Sullivan P.C.
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