Regions of papilloma virus E1 helicase involved in E1 oligomerization

Abstract

There is provided an amino acid sequence comprised within the PV E1 protein region A delineated by amino acids 352 and 439, and any derivative variant or fragment thereof, necessary for the oligomerization of the E1 protein. This amino acid sequence is capable of self-association and of associating with the full length E1 protein and any derivative, variant or fragment thereof comprising the sequence of this invention. A specific aspect of this first embodiment, the amino acid domain of this invention delimited by amino acids 353 to 438 of the PV E1 protein. More particularly, the amino acid domain of this invention is as defined by SEQ ID NO. 2. There is also provided a cross-linking assay to directly measure the level of oligomerization (or inhibition thereof) of the E1 protein. In accordance with a fourth embodiment of this invention, there is provided a N-terminally truncated E1 protein. More particularly, one aspect of this fourth embodiment encompasses the E1 protein delimited by amino acid 72 to 649 (SEQ ID NO. 78).

1 . An amino acid sequence within a papilloma virus E1 protein region A necessary for oligomerization of an E1 protein comprising, amino acid residues delineated by amino acids 352-439 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         2 . The amino acid sequence of  claim 1 , wherein said sequence is further delineated by amino acids 352-432 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         3 . The amino acid sequence of  claim 2 , wherein said sequence is further delineated by amino acids 352-417 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         4 . The amino acid sequence of  claim 1 , wherein said sequence is delimited by amino acids 353 to 438 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         5 . The amino acid sequence of  claim 2;  wherein said sequence is delimited by amino acids 353 to 431 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         6 . The amino acid sequence of  claim 3 , wherein said sequence is delimited by amino acids 353 to 416 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         7 . An N-terminally truncated E1 protein capable of self-association and of associating with a full-length E1 protein.  
     
     
         8 . The E1 protein of  claim 7 , wherein said E1 protein has about its first 70 N-terminal amino acids deleted.  
     
     
         9 . The E1 protein of  claim 8 , wherien said protein is delimited by amino acids 72-649 as numbered from an E1 protein from HPV-11, and any variant, derivative or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         10 . An amino acid sequence defined by SEQ ID NO. 2 and any derivative, variant or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         11 . An amino acid sequence defined by SEQ ID NO. 3 and any derivative, variant or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         12 . An amino acid sequence defined by SEQ ID NO. 4 and any derivative, variant or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         13 . An amino acid sequence defined by SEQ ID NO. 78 and any derivative, variant or fragment thereof capable of self-association and of associating with a full-length E1 protein.  
     
     
         14 . The amino acid of  claim 1 , wherein said sequence originates from a virus selected from the group consisting of papilloma viruses, SV40 and polyoma virus.  
     
     
         15 . The amino acid sequence of  claim 14 , wherein said sequence from said papillomavirus E1 protein is from a cottontail rabbit papillomavirus (CRPV), bovine papillomavirus (BPV), or human papillomavirus (HPV).  
     
     
         16 . The amino acid sequence of  claim 15 , wherein said HPV E1 protein is from an HPV low risk or an HPV high risk type.  
     
     
         17 . The sequence of  claim 16 , wherein said HPV E1 protein is from said low risk HPV type selected from the group consisting of types 6, 11, and 13.  
     
     
         18 . The sequence of  claim 16 , wherein said HPV E1 protein is from said high risk HPV type selected from the group consisting of types 16, 18, 31, 33, 35, 42, 52, or 58.  
     
     
         19 . The sequence of  claim 17 , wherein said HPV E1 protein is from said low risk HPV type 11.  
     
     
         20 . The sequence of  claim 18 , wherein said HPV E1 protein is from said high risk HPV type 16.  
     
     
         21 . A method for treating controlling, preventing or eliminating papillomavirus infection in a mammal comprising administering an effective amount of the amino acid sequence according to  claim 1 ,  2 , or  3  to the mammal.  
     
     
         22 . A cross-linking assay to evaluate the level of oligomerization of the E1 protein, said assay comprising the steps of: 
 a. combining labeled-E1 protein selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 78; and any derivative, variant or fragment thereof capable of self-association and of associating with a full-length E1 protein, with a DNA fragment, and incubating for a period of time, to allow the E1 protein and the DNA to form a complex,    b. cross-linking the E1 protein and the DNA in the complex with a cross-linking agent,    c. isolating the E1 protein/DNA complex from the non-complexed DNA; and    d. separating the E1 protein electrophoretically such that the migration of E1 on a gel is an indication of the level of oligomerization of E1.    
     
     
         23 . The assay of  claim 22 , wherein said DNA fragment is a single stranded DNA.  
     
     
         24 . The assay of  claim 22 , wherein said E1-DNA complex is isolated from the free DNA by column chromatography, centrifugation, extraction, filtration, immunoprecipitation or immobilizing on solid support using an antibody directed against the E1 protein.  
     
     
         25 . The assay of  claim 24 , wherein said E1 is immunoprecipitated using a polyclonal antibody.  
     
     
         26 . The assay according to  claim 22 , wherein said E1 protein is labeled with a radio isotope.  
     
     
         27 . The assay according to  claim 26 , wherein said E1 is labaled with  35 S and is detected on said gel by radio-imaging techniques.  
     
     
         28 . The assay according to  claim 22 , wherein said E1 wherein said cross-linking agent is bismaleimidohexane (BMH).  
     
     
         29 . The assay of  claim 22 , wherein said E1 protein is obtained by coupled transcription/translation synthesis in a rabbit reticulocyte lysate or is made by recombinant technology.