Title:  Self-replicating RNA molecule from hepatitis C virus

United States Patent:  6,706,874

Issued:  March 16, 2004

Inventors:  Kukolj; George (2100 Cunard Street, Laval, Quebec, CA H7S 2G5); Pause; Arnim (2100 Cunard Street, Laval, Quebec, CA H7S 2G5)

Appl. No.:  029907

Filed:  December 21, 2001

Abstract

A unique HCV RNA molecule is provided having an enhanced efficiency of establishing cell culture replication. Novel adaptive mutations have been identified within the HCV non-structural region that improves the efficiency of establishing persistently replicating HCV RNA in cell culture. This self-replicating polynucleotide molecule contains, contrary to all previous reports, a 5'-NTR that can be either an A as an alternative to the G already disclosed and therefore provides an alternative to existing systems comprising a self-replicating HCV RNA molecule. The G.fwdarw.A mutation gives rise to HCV RNA molecules that, in conjunction with mutations in the HCV non-structural region, such as the G(2042)C/R mutations, possess greater efficiency of transduction and/or replication. These RNA molecules when transfected in a cell line are useful for evaluating potential inhibitors of HCV replication.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a 5'-non translated region of the hepatitis C virus wherein its highly conserved guanine at position 1 is substituted for adenine.

Particularly, the present invention provides a hepatitis C virus polynucleotide comprising adenine at position 1 as numbered according to the 1377/NS2-3' construct (Lohmann et al. 1999, Science 285, 110-113, Accession # AJ242651).

Particularly, the invention provides a HCV self-replicating polynucleotide comprising a 5'-terminus consisting of ACCAGC (SEQ ID NO. 8).

In a second embodiment, the present invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising one or more amino acid substitution selected from the group consisting of: R(1135)K; S(1148)G; S(1560)G; K(1691)R; L(1701)F; I(1984)V; T(1993)A; G(2042)C; G(2042)R; S(2404)P; L(2155)P; P(2166)L and M(2992)T.

Particularly, the invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising the any one of the amino acid substitutions as described above, further comprising the amino acid substitution E(1202)G.

More particularly, the invention provides a HCV self-replicating polynucleotide encoding a polyprotein comprising a G2042C or a G2042R mutation.

Most particularly, the invention provides for HCV self-replicating polynucleotide comprising a nucleotide substitution G.fwdarw.A at position 1, and said polynucleotide encodes a polyprotein further comprising a G2042C or a G2042R mutation. encodes a polyprotein further comprising a G2042C or a G2042R mutation.

Particularly, the polynucleotide of the present invention can be in the form of RNA or DNA that can be transcribed to RNA.

In a third embodiment, the invention also provides for an expression vector comprising a DNA form of the above polynucleotide, operably linked with a promoter.

According to a fourth embodiment, there is provided a host cell transfected with the self-replicating polynucleotide or the vector as described above.

In a fifth embodiment, the present invention provides a RNA replication assay comprising the steps of:

incubating the host cell as described above in the absence or presence of a potential hepatitis C virus inhibitor;

isolating the total cellular RNA from the cells;

analyzing the RNA so as to measure the amount of HCV RNA replicated;

comparing the levels of HCV RNA in cells in the absence and presence of the inhibitor.

In a sixth embodiment, the invention is directed to a method for testing a compound for inhibiting HCV replication, including the steps of:

a) treating the above described host cell with the compound;

b) evaluating the treated host cell for reduced replication, wherein reduced replication indicates the ability of the compound to inhibit replication.

Preferred Embodiments

Particularly, the invention provides a HCV self-replicating polynucleotide molecule comprising a 5'-terminus consisting of ACCAGC (SEQ ID NO. 8).

According to the first embodiment of this invention, there is particularly provided a HCV polynucleotide construct comprising:

a 5'-non translated region (NTR) comprising the sequence ACCAGC at, or proximal to, its 5'-terminus;

a HCV polyprotein coding region; and

a 3'-NTR region.

In a second embodiment, the present invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising one or more amino acid substitution selected from the group consisting of: R(1135)K; S(1148)G; S(1560)G; K(1691)R; L(1701)F; I(1984)V; T(1993)A; G(2042)C; G(2042)R; S(2404)P; L(2155)P; P(2166)L and M(2992)T.

Particularly, the invention is directed to a HCV self-replicating polynucleotide encoding a polyprotein comprising the any one of the amino acid substitutions as described above, further comprising the amino acid substitution E(1202)G.

Alternatively, the first embodiment of the present invention is directed to HCV self-replicating polynucleotide molecule comprising a G2042C/R mutation.

According to the second embodiment, the present invention particularly provides a HCV polynucleotide construct comprising:

a 5'-NTR region comprising the sequence ACCAGC at, or proximal to, its 5'-terminus;

a HCV polyprotein region coding for a HCV polyprotein comprising a G(2042)C or a G(2042)R mutation; and

a 3'-NTR region.

Preferably, the polynucleotide construct of the present invention is a DNA or RNA molecule. More preferably, the construct is a RNA molecule. Most preferably, the construct is a DNA molecule.

More particularly, the first embodiment of this invention is directed to a RNA molecule encoded by the DNA molecule selected from the group consisting of: SEQ ID NO.2,4,5,6,7,24 and 25.

Most particularly, the invention provides a DNA molecule selected from the group consisting of: SEQ ID NO. 2, 4, 5, 6, 7, 24 and 25.

In a third embodiment, the invention also is directed to an expression vector comprising DNA forms of the above polynucleotide, operably linked with a promoter.

Preferably, the promoter is selected from the group consisting of: T3, T7 and SP6.

According to a fourth embodiment, there is provided a host cell transfected with the self-replicating polynucleotide or vector as described above. Particularly, the host cell is a eukaryotic cell line. More particularly, the eukaryotic cell line is a hepatic cell line. Most particularly, the hepatic cell line is Huh-7.

In a fifth embodiment, the present invention provides a RNA replication assay comprising the steps of:

a) incubating the host cell as described above under conditions suitable for RNA replication;

b) isolating the total cellular RNA from the cells; and

c) analyzing the RNA so as to measure the amount of HCV RNA replicated.

Preferably, the analysis of RNA levels in step c) is carried out by amplifying the RNA by real-time RT-PCR analysis using HCV specific primers so as to measure the amount of HCV RNA replicated.

Alternatively in this fifth embodiment, the construct comprises a reporter gene, and the analysis of RNA levels in step c) is carried out by assessing the level of reporter expressed.

According to a preferred aspect of the sixth embodiment, the invention is directed to a method for testing a compound for inhibiting HCV replication, including the steps of:

a) carrying step a) as described in the above assay, in the presence or absence of the compound;

b) isolating the total cellular RNA from the cells; and

c) analyzing the RNA so as to measure the amount of HCV RNA replicated.

d) comparing the levels of HCV RNA in cells in the absence and presence of the inhibitor,

wherein reduced RNA levels is indicative of the ability of the compound to inhibit replication.

Preferably, the cell line is incubated with the test compound for about 3-4 days at a temperature of about 37^oC.

Claim 1 of 10 Claims

What is claimed is:

1. A HCV self-replicating polynucleotide comprising:

(a) a 5'-Non Translated Region consisting of ACCAGC (SEQ ID NO. 8);

(b) a HCV polynucleotide coding region encoding a HCV polyprotein comprising: NS3, NS4A, NS4B, NS5A, and NS5B proteins; and

(c) a 3'-Non Translated Region.

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