Carboxy-terminal five amino acids of the nucleocapsid protein of vesicular stomatitis virus are required for encapsidation and replication of genome RNA.
The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.
Das
T
T
The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Chakrabarti
B K
BK
Chattopadhyay
D
D
Banerjee
A K
AK
D000596:Amino Acids / Q000235:genetics; D000818:Animals; D016679:Genome, Viral; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D011817:Rabbits; D017421:Sequence Analysis; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D000596:Amino Acids; D012367:RNA, Viral