Viral Particle-Like Construct and Method of Forming the Same Under Physiological Conditions

Abstract

There is provided a novel method of forming uniform viral particles under physiological conditions. The method of forming uniform-sized viral particle aggregates composed of viral protein is characterized by incubating a viral protein such as SV40 virus VP 1 at pH 5.0 to 7.0, room temperature, in the presence of 130 mM to 170 mM sodium chloride and 1.5 mM to 2.5 mM divalent cation, and in the presence of a particle formation acceleration factor such as SV40 VP2. For encapsulation of a substance to be encapsulated in the viral particles, the substance to be encapsulated is included during the incubation.

1 . A uniform-sized viral particle-like structure composed of a viral protein and a particle formation acceleration factor. 
     
     
         2 . A uniform-sized viral particle-like structure composed of a viral protein and a particle formation acceleration factor, which houses a substance to be encapsulated. 
     
     
         3 . A viral particle-like structure according to  claim 1 , wherein the viral protein is VP1 capsid protein of SV40 virus, JC virus or BK virus. 
     
     
         4 . A viral particle-like structure according to  claim 3 , wherein the SV40 virus protein is VP1 capsid protein or a mutant thereof. 
     
     
         5 . A viral particle-like structure according to  claim 4 , wherein the VP1 capsid protein mutant is a protein which is VP1 capsid protein having the amino acid sequence listed as SEQ ID NO: 2 with a deletion, addition or amino acid substitution of one or more amino acids. 
     
     
         6 . A viral particle-like structure according to  claim 5 , wherein the substitution is at least one amino acid substitution from among Glu at position 49, Glu at position 51, Glu at position 160, Glu at position 163, Ser at position 216, Lys at position 217, Glu at position 219, Glu at position 332, Glu at position 333 and Asp at position 348 of the amino acid sequence listed as SEQ ID NO: 2. 
     
     
         7 . A viral particle-like structure according to any one of  claim 1 , wherein the particle formation acceleration factor is the viral particle capsid protein, the N-terminal region of the protein having particle formation accelerating activity, or a protein which is modified with a deletion, addition and/or amino acid substitution of one or more amino acids of the protein and which retains particle formation accelerating activity. 
     
     
         8 . A viral particle-like structure according to  claim 7 , wherein the viral particle capsid protein is the capsid protein VP2 of SV40 virus, JC virus or BK virus. 
     
     
         9 . A viral particle-like structure according to  claim 7 , wherein the viral particle capsid protein is the SV40 viral capsid protein VP2 having the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         10 . A viral particle-like structure according to  claim 7 , wherein the viral particle capsid protein is a portion of the capsid protein VP2 of SV40 virus comprising at least amino acids 1 to 272 of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         11 . A viral particle-like structure according to  claim 7 , wherein the viral particle capsid protein is a portion of the capsid protein VP2 of SV40 virus comprising at least amino acids 1 to 58, or 59 to 118, or  119  to  152 , or  153  to  272  of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         12 . A viral particle-like structure according to  claim 7 , wherein the viral particle capsid protein is a portion of the capsid protein VP2 of SV40 virus comprising at least the amino acid sequence of the VP2-binding region from residues 273 to 307 of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         13 . A viral particle-like structure according to  claim 2 , wherein the substance to be encapsulated is a bioactive substance or non-bioactive substance, or a mixture thereof. 
     
     
         14 . A viral particle-like structure according to  claim 2 , wherein the non-bioactive substance is a low molecular substance, a high molecular substance or a mixture thereof. 
     
     
         15 . A viral particle-like structure according to  claim 13 , wherein the bioactive substance is a nucleic acid, protein or low molecular substance. 
     
     
         16 . A method of forming uniform-sized viral particle aggregates composed of viral protein and a particle formation acceleration factor, characterized by incubating viral protein at pH 5 to 10 at room temperature, in the presence of 130 mM to 500 mM monovalent cation and 2 mM to 50 mM divalent cation, and in the presence of a particle formation acceleration factor. 
     
     
         17 . A method of forming uniform-sized viral particle aggregates composed of a substance to be encapsulated, and a viral protein and a particle formation acceleration factor which surrounds it, characterized by incubating viral protein and the substance to be encapsulated at pH 5 to 10 at room temperature, in the presence of 130 mM to 500 mM monovalent cation and 2 mM to 50 mM divalent cation, and in the presence of a particle formation acceleration factor. 
     
     
         18 . A method according to  claim 16 , wherein the monovalent cation is sodium ion. 
     
     
         19 . A method according to  claim 16 , wherein the divalent cation is calcium ion. 
     
     
         20 . A method according to  claim 16 , wherein the concentration of the monovalent cation is 150 mM, and the concentration of the divalent cation is 2 mM. 
     
     
         21 . A method according to  claim 16 , wherein the viral protein is the VP1 capsid protein of SV40 virus, JC virus or BK virus. 
     
     
         22 . A method according to  claim 16 , wherein the particle formation acceleration factor is the viral particle capsid protein, the N-terminal region of the protein having particle formation accelerating activity, or a protein which is modified with a deletion, addition and/or amino acid substitution of one or more amino acids of the protein and which retains particle formation accelerating activity. 
     
     
         23 . A method according to  claim 22 , wherein the viral particle capsid protein is the capsid protein VP2 of SV40 virus, JC virus or BK virus. 
     
     
         24 . A method according to  claim 22 , wherein the viral particle capsid protein is SV40 viral capsid protein VP2 having the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         25 . A method according to  claim 23 , wherein the viral particle capsid protein is a portion of the capsid protein VP2 of SV40 virus comprising at least amino acids 1 to 272 of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         26 . A method according to  claim 23 , wherein the viral particle capsid protein is a portion of the capsid protein VP2 of SV40 virus comprising at least amino acids 1 to 58, or 59 to 118, or 119 to 152, or 153 to 272 of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         27 . A method according to  claim 17 , wherein the substance to be encapsulated is a bioactive substance or non-bioactive substance, or a mixture thereof. 
     
     
         28 . A method according to any  claim 17 , wherein the non-bioactive substance is a low molecular substance, a high molecular substance or a mixture thereof. 
     
     
         29 . A method according to  claim 27 , wherein the bioactive substance is a nucleic acid, protein or low molecular substance. 
     
     
         30 . A method of introducing a bioactive substance into virus-like particles composed of viral protein, characterized by coexpressing in host cells the viral protein and capsid protein VP2 or a portion thereof comprising the binding region of the viral protein and having a bioactive substance linked thereto. 
     
     
         31 . A method according to  claim 30 , wherein the viral protein is SV40 virus VP1 capsid protein or a mutant thereof, and the VP2 protein is a portion of SV40 virus capsid protein VP2 comprising at least the amino acid sequence of the VP1-binding region from positions 273 to 307 of the amino acid sequence listed as SEQ ID NO: 1. 
     
     
         32 . A composition for introduction of a bioactive substance into cells, whose active component is a viral particle structure according to  claim 13 . 
     
     
         33 . A method for producing viral particle aggregates composed of viral protein encapsulating a polymer with a negatively charged surface, the method being characterized by mixing the viral protein with the polymer at 0.01 to 100 parts (by weight) with respect to 360 parts of the viral capsid protein, and dialyzing the mixture against an aqueous solution containing a monovalent metal salt and a divalent metal salt. 
     
     
         34 . A method according to  claim 33 , wherein the negatively charged polymer is DNA, RNA or a synthetic nucleic acid-like structure. 
     
     
         35 . A method according to  claim 33 , wherein in the method for producing the viral particle aggregates, the weight ratio of the viral protein and the negatively charged polymer added to 360 parts of the viral capsid protein is 0.2 or greater. 
     
     
         36 . A method according to  claim 33 , wherein the viral protein is the VP1 capsid protein of SV40 virus, JC virus or BK virus. 
     
     
         37 . A method according to  claim 36 , wherein the viral protein is the SV40 virus VP1 capsid protein represented by SEQ ID NO: 2, or a mutant thereof. 
     
     
         38 . A method according to  claim 33 , wherein the monovalent metal ion is sodium ion, and the divalent metal ion is calcium ion.