Modified polymerases and attenuated viruses and methods of use thereof
AssigneeThe Penn State Research Foundation
InventorsCraig E. Cameron, Christian Castro, Jamie J. Arnold
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Abstract
The invention encompasses compositions and methods relating to viral polymerases having one or more substitutions of different amino acids at conserved regions of the polymerase yields enzymes with varying rates and fidelity of replication. A universally applicable, polymerase-mechanism-based strategy for production of attenuated viruses and anti-viral vaccines is disclosed.
Claims19
An attenuated virus comprising a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases,
- wherein the substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification.
A vaccine comprising:
- (a) an attenuated virus comprising a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases,
- wherein the substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification;
- and
- (b) a pharmaceutically acceptable carrier.
A purified nucleic acid comprising a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases,
- wherein the substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification.
A method of attenuating a virus comprising modifying a viral polymerase gene that encodes a polymerase, such that the modification results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases,
- wherein the substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification.
Dependencies are inferred from the claim text.