Analysis For Gene Functions And Expression Strategy Of Beet Black Scorch Virus

Based on the full-length infectious cDNA pUBF52 of Beet black scorch virus (BBSV), reverse genetic analysis was carried for gene functions and expression strategy of BBSV by means of in vitro transcription of mutant viral RNAs, GFP labeled expression. By mutation of nucleotide substitution, frame-shift and deletion, it was proven that the P22 and P82 at the 5' proximal end of BBSV genome were both involved into its replicase (RdRp) essentially. By site-directed mutagenesis of translational start codons or premature, the four small ORFs including P5, P7a, P7b and P5' that are centrally located in the viral genome by computer prediction were analyzed for their functions during BBSV infection. The results showed that, except the P5, other three ORFs of P7a, P7b and P5' were coordinately responsible for the local lesion symptoms and viral RNA accumulation after BBSV infection of Chenopodium amaranticolor.For fluorescent expression in plant cells, a GFP gene was fused BBSV RNAs as a substitute of the C? in the each mutant of P7a, P7b and P5'. In contrast to the infection by wild type BBSV labeled with GFP from pBGFP, in which the GFP was observed both on nuclei and cell membranes in the cells around the initially infected cell, GFP expressions of mutants P7a, P7b and P5' were limited in the nuclei of the original infected cell. Combining with the previous report on BBSV coat protein gene, these results indicated that total six proteins were possibly encoded by BBSV genome, including the RdRp components of P22 and P82, the P7a, P7b and P5' proteins associated with BBSV movement from cell-to-cell, and the P24 coat protein, among which the P5' is reported first time in Necroviruses.In further investigation, a strategy for the mRNA expression of BBSV multi-cistron genome was revealed with two subgenomic RNA (sgRNA) in BBSV particles or C. amaranticolor infected by the in vitro transcripts. By 5' -RACE and mutagenesis of infectious cDNAs, the transcription start sites of sgRNAl and sgRNA2 were determined as G²²⁰⁹ or G²⁵²⁶, respectively. So that, the sgRNAl is responsible for protein expressions of P7a, P7b and P5' and the sgRNA2 is for the coat protein.Mutations of nucleotides from position 2204 to 2213 nt around G²²⁰⁹ and 2521 to 2530 nt around G²⁵²⁶ showed that mutation of C²²⁰⁶, or any of C²⁵²¹, C²⁵²³ and C²⁵²⁴ would abolish the form of sgRNAl or sgRNA2, respectively. The following mutations of G which complement with these C in the second structure of BBSV and corresponding base pair showed that the base pairing is more important than these C themselves. According to the secondary structure of BBSV RNA by computer analysis, the results from mutations of the base-pairings of C²²⁰⁶-G²¹¹⁸, C²⁵²¹-G²³⁸⁰, C²⁵²³-G²³⁷⁸ and C²⁵²⁴-G²³⁷⁷ indicated that the predicted stem-loop structures upstream from the two transcription start sites played important roles on the RNA transcription. The nucleotide deletions around the sgRNAs start sites provided the further evidences that the promoter region for the sgRNA2 perhaps ranged from 2339 nt to 2558 nt as a stable stem-loop complex form. Since a similar structure also was found in the sgRNAl, itwas speculated that the mechanism of premature termination was employed in transcription of BBSV sgRNAs.Similar to other Necroviruses, the stem-loop structures involved in 5' -3' RNA-RNA interaction were also found in the proximal ends of either BBSV genomic RNA or the subgenomic RNAs, in which the nucleotide sequences in the loop regions were highly conserved and reversely complementary. In order to know the possible interaction between these stem-loop structures, mutagenesis analysis was carried out. The preliminary results showed that the conservation of nucleotide sequence in the loop region and the base-pairing in the stem of the stem-loop structures were necessary to keep the correct RNA folding essential for the virus replication.