Peptide aptamers that bind to the rep proteins of ssdna viruses

Abstract

Polypeptides and fusion proteins that bind to eukaryotic viruses, in particular, eukaryotic single-stranded DNA (ssDNA) viruses are provided. The polypeptides and fusion proteins bind to the replication initiation proteins (Rep) of ssDNA viruses and optionally inhibit viral replication and/or viral infection. The virus can be a plant pathogen or animal pathogen. Consensus sequences used to identify polypeptides that bind to eukaryotic viruses are also provided.

1 - 27 . (canceled) 
     
     
         28 . A polypeptide comprising an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:89;   (b) a fragment of the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:89, wherein the fragment comprises MHXXXXXG or (L/M)GGXXP; and wherein the fragment binds to a viral replication (Rep) protein; and   (c) an amino acid sequence that is at least 80% similar to the amino acid sequence of (a) or (b) and binds to a viral Rep protein.   
     
     
         29 . The polypeptide according to  claim 28 , wherein the polypeptide binds to a Rep protein selected from the group consisting of a geminivirus Rep protein, a nanovirus Rep protein, a circovirus Rep protein and combinations thereof. 
     
     
         30 . The polypeptide according to  claim 28 , wherein the polypeptide binds to a geminivirus Rep protein. 
     
     
         31 . The polypeptide according to  claim 28 , wherein the polypeptide binds to a Rep protein selected from the group consisting of a tomato golden mosaic virus (TGMV) Rep protein, a cabbage leaf curl virus (CbLCV) Rep protein, a tomato yellow leaf curl virus (TYLCV) Rep protein, a tomato mottle virus (ToMoV) Rep protein, an African cassava mosaic virus (ACMV) Rep protein, a maize streak virus (MSV) Rep protein, and a cotton leaf curl virus (CLCuV) Rep protein, and combinations thereof. 
     
     
         32 . The polypeptide according to  claim 28 , wherein the polypeptide comprises an amino acid sequence that is at least 95% similar to the amino acid sequence of (a) and binds to a viral Rep protein. 
     
     
         33 . The polypeptide according to  claim 32 , wherein the amino acid sequence comprises MHXXXXXG. 
     
     
         34 . The polypeptide according to  claim 32 , wherein the amino acid sequence comprises (L/M)GGXXP. 
     
     
         35 . A fusion protein comprising the polypeptide according to  claim 28  optionally comprising thioredoxin. 
     
     
         36 . The polypeptide according to  claim 28 , wherein the polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:89, wherein the fragment comprises (L/M)GGXXP, and wherein the fragment binds to a viral Rep protein. 
     
     
         37 . The polypeptide according to  claim 28 , wherein the polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:55, wherein the fragment comprises MHXXXXXG, and wherein the fragment binds to a viral Rep protein. 
     
     
         38 . The polypeptide according to  claim 28 , wherein the amino acid sequence comprises no more than three amino acid substitutions, insertions and/or deletions as compared with the amino acid sequence of (a). 
     
     
         39 . The polypeptide according to  claim 28 , wherein the amino acid sequence comprises no more than two amino acid substitutions as compared with the amino acid sequence of (a), and wherein all of the amino acid substitutions are conservative substitutions. 
     
     
         40 . A method of detecting a viral infection, the method comprising:
 (a) contacting a sample with a polypeptide according to  claim 28 ; and   (b) detecting the presence or absence of binding between the polypeptide and a target, wherein the binding of the polypeptide to the target in the sample indicates the presence of a virus.   
     
     
         41 . The method of  claim 40 , wherein the target is a viral Rep protein. 
     
     
         42 . A method of detecting a viral infection, the method comprising:
 (a) contacting a sample with a fusion protein according to  claim 35 ; and   (b) detecting the presence or absence of binding between the polypeptide and a target, wherein the binding of the polypeptide to the target in the sample indicates the presence of a virus.   
     
     
         43 . A method of inhibiting viral replication in a plant cell, the method comprising introducing the polypeptide of  claim 28  into the plant cell in an amount effective to inhibit viral replication. 
     
     
         44 . The method of  claim 43 , wherein introducing the polypeptide of  claim 28  comprises introducing an isolated nucleic acid encoding an amino acid sequence of SEQ ID NO:55 or SEQ ID NO:89, or a fragment thereof, wherein the fragment comprises MHXXXXXG or (L/M)GGXXP. 
     
     
         45 . A method of inhibiting viral replication in a plant cell, the method comprising introducing the fusion protein of  claim 35  into the plant cell in an amount effective to inhibit viral replication. 
     
     
         46 . A method of providing increased resistance to viral infection in a plant, the method comprising introducing the polypeptide of  claim 28  into the plant in an amount effective to increase resistance to viral infection. 
     
     
         47 . The method of  claim 46 , wherein introducing the polypeptide of  claim 28  comprises introducing an isolated nucleic acid encoding an amino acid sequence of SEQ ID NO:55 or SEQ ID NO:89, or a fragment thereof, wherein the fragment comprises MHXXXXXG or (L/M)GGXXP. 
     
     
         48 . A method of providing increased resistance to viral infection in a plant, the method comprising introducing the fusion protein of  claim 35  into the plant in an amount effective to increase resistance to viral infection. 
     
     
         49 . A method of making a transgenic plant having increased resistance to a virus infection, the method comprising introducing an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of  claim 28  into a plant, thereby producing a transgenic plant having increased resistance to infection by a virus. 
     
     
         50 . A method of making a transgenic plant having increased resistance to a virus infection, the method comprising:
 transforming a plant cell with an isolated nucleic acid encoding the polypeptide of  claim 28 ; and   regenerating a transgenic plant from said transformed plant cell, thereby producing a transgenic plant having an increased resistance to infection by a virus.   
     
     
         51 . A transgenic plant comprising an isolated nucleic acid encoding the polypeptide of  claim 28 . 
     
     
         52 . The transgenic plant of  claim 51 , wherein the polypeptide binds to a Rep protein selected from the group consisting of a geminivirus Rep protein, a nanovirus Rep protein, a circovirus Rep protein and combinations thereof. 
     
     
         53 . The transgenic plant of  claim 51 , wherein the polypeptide binds to a geminivirus Rep protein. 
     
     
         54 . The transgenic plant of  claim 51 , wherein the amino acid sequence comprises no more than three amino acid substitutions, insertions and/or deletions as compared with the amino acid sequence of (a). 
     
     
         55 . The transgenic plant of  claim 51 , wherein the amino acid sequence comprises no more than two amino acid substitutions as compared with the amino acid sequence of (a), and wherein all of the amino acid substitutions are conservative substitutions.