Structural and functional properties of the vesicular stomatitis virus nucleoprotein-RNA complex as revealed by proteolytic digestion.
To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid-RNA complex (vN-RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN-RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN-RNA complex (vDeltaN-RNA) carrying N-terminal 336 amino acids of the N protein (DeltaN) was inactive in transcription. The DeltaN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN-RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N-RNA complex purified from bacteria (bN-RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN-RNA and vDeltaN-RNA are discussed.
Sarkar
Anindya
A
Department of Molecular Genetics, Section of Virology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Chattopadhyay
Santanu
S
Cox
Robert
R
Luo
Ming
M
Banerjee
Amiya K
AK
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002918:Chymotrypsin / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000737:chemistry / Q000378:metabolism
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012367:RNA, Viral; D015678:Viral Structural Proteins; D002918:Chymotrypsin