Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-25
2001-06-19
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S194000
Reexamination Certificate
active
06248589
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 9.4 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) 2
nd 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.
SUMMARY OF THE INVENTION
The present invention provides a recombinant protein of HCV having RDRP activity (r-HCV-RDRP) obtainable by expression in a host mammalian or bacterial cell of a modified NS5B coding region of HCV. The modification includes addition at the amino terminus of a methionine residue and optionally from 1-20 additional amino acids interposed between the N-terminal methionine and the N-terminal serine of unmodified NS5B gene product. The modification also includes deletion at the amino terminus of up to 9 amino acids to provide an amino-terminal methionine. Two methionines occur naturally according to the deduced sequence of wild-type HCV-RDRP. Therefore, modification includes deletion to remove amino acids lying N-terminal to either methionine or, alternatively, deletion to some intermediate point between the two methionines plus addition of an N-terminal methionine codon. A combination of deletions and insertions, within the limits described is also contemplated. Added amino acid sequence can be devised to create a specific protease cleavage site to permit post translational modification of the recombinant HCV-RDRP expression produce, in vivo or in vitro. Such post-transcriptional modification can be used to generate exactly the amino acid sequence encoded by NS5B, having an N-terminal serine. Added amino acid sequence can be devised to generate an affinity ligand binding site, for convenience and ease of purification. The data reported herein were obtained with a r-HCV-RDRP having an N-terminal MA (Met-Ala) dipeptide, giving an N-terminal sequence MASMSY (SEQ ID NO:6) instead of the predicted SMSY sequence of the wild-type processed protein. The coding sequence of NS5B is accordingly modified to include a met codon (ATG) at the 5′-end, as well as, optionally, codons for other amino acids to be included or deleted. Minimal modifications are preferred, in order to avoid potential deleterious effects on enzyme activity, and to avoid creating artificial epitopes. The r-HCV-RDRP can be expressed in
E. coli
and in mammalian cells to yield active RDRP. The expression of active r-HCV-RDRP in
E. coli
demonstrates that no other HCV-encoded protein is necessary for polymerase activity.
The invention further provides r-HCV-RDRP in solubilized form, and a method of solubilization without destroying activity.
The invention also provides methods for purifying solubilized HCV-RDRP. One such method, to be used in combination with others, is affinity chromatography, using antibody to r-HCV-RDRP as the affinity ligand. Other affinity ligands are obtained by a combinatorial library approach as described, e.g., by Wu, J. et al. (1994)
Biochemistry
33:14825-14833; and Ohlmeyer, M. H. J. et al. (1993)
Procl. Nat. Acad. Sci. USA
90:10922-10926.
In addition, the invention provides polyclonal or monoclonal antibodies specific for HCV-RDRP. Such antibodies can be made by known techniques, using the purified enzyme as antigen. Such antibodies bind either r-HCV-RDRP or wild-type HCV-RDRP. The availability of such antibodies makes it possible to prepare an affinity-labeled chromatography matrix for rapid purification of HCV-RDRP. The antibody also makes possible rapid detection of HCV-RDRP in biological materials, for example, in serum of HCV-infected patients.
The invention further provides a method for transfecting a mammalian cell with HCV-RDRP and expressing the enzyme within the cell. Consequently, the invention also provides a transfected mammalian cell line expressing r-HCV-RDRP. Such cells are useful mammalian cell line expressing r-HCV-RDRP. Such cells are useful for assaying the effects of candidate anti-viral compounds as inhibitors of RDRP activity.
Therefore, the invention also provides a method for screening possible inhibitors of RDRP activity in vivo. Compounds with inhibitory activity can have anti-viral activity, since inhibition of the polymerase inhibits viral replication and expression of virus gene products. The in vitro assay is advantageous because it can rule out compounds which cannot enter the infected cell. One class of attractive candidate compounds is the nucleoside analogs; compounds which after being modified (phosphorylated) within cells can bind to substrate sites on the enzyme or which can be incorporated into a newly synthesized RNA but whose presence there disrupts normal function of the HCV polymerase or further replication of an RNA containing the analog. Acyclovir is one example of a very effective
Al Reinoldus H.
Hagedorn Curt H.
Emory University
Greenlee Winner and Sullivan PC
Patterson Jr. Charles L.
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