Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

Abstract

Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

Fig. 1

Amino acid sequence alignment of the AmCPV S2 encoded RdRp with that of polio virus (PDB code 1RDR), RHDV (PDB code 1KHV), reovirus (PDB code 1N35), HCV (PDB Code 1QUV). Conserved motifs are highlighted with box and marked below. The alignment is done to show the equivalent position of the different signature motifs.

Fig. 2

Phylogenetic analysis of AmCPV RdRP with other members of the Reoviridae family by neighbor joining method. The number at each node represents bootstrap value of 100 replicates. GenBank accession numbers are shown in the parenthesis.

Fig. 3

(A) Analysis of E. coli expressed AmCPV RdRp by SDS-PAGE. Lane M, Molecular weight marker; lane 1, uninduced E. coli cell lysate; lane 2, induced E. coli cell lysate; lane 3, Ni-NTA purified AmCPV RdRp. (B) Western blot analysis of wild and mutant AmCPV RdRp expressed in insect cells. Lane M, molecular weight marker; lane 1, purified wild type AmCPV RdRp; lane 2, purified GAD mutant protein; lane 3, purified GAA mutant protein. (Right panel western blot, left panel SDS-8% PAGE).

Fig. 4

(A). RdRp assay with different concentration of AmCPV S2 encoded protein. The reactions were performed at different concentrations of purified protein with fixed concentration of Oligo U₍₁₈₎ primer and Poly (A) template. Incorporation of [α³²P] UTP in fmol is indicated as mean ± standard deviation of three independent experiments. Another experiment was done using in vitro transcribed 3' positive strand AmCPV S2 RNA as template and the reaction products were analyzed through 1% formaldehyde agarose gel and autoradiographed (insets). Lanes 1 to 6 represent reaction products with 0.1 µM, 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM and 0.8 µM purified AmCPV S2 encoded protein, respectively. (B) RdRp assay of AmCPV S2 encoded protein (0.4 µM) with different concentration (0.02–0.22 µM) of template.

Fig. 5

Effect of divalent metal ions, pH, temperature, salts and incubation time on the RdRp activity. The reactions were carried out (A) at different concentrations of Mg⁺⁺ and Mn⁺⁺ or (B) at different pH or (C) at different time and temperature or (D) at different concentration of KCl or NaCl for the determination of maximum RdRp activity using poly (A) RNA as template (175 ng) and oligo U₍₁₈₎ primer (50 ng) in presence of 0.4 µM AmCPV S2 encoded protein. Values are mean ± standard deviation of three independent experiments.

Fig. 6

(A) Schematic diagram of the positions of three pairs of primers that were used to generate a promoter (T7 or SP6) containing cDNA fragments by PCR. The amplified cDNA fragments of AmCPV were then used to produce the 5′ positive strand RNA (277 nt), the 3′ positive strand (204 nt), and 3′ negative strand RNA (245 nt) by in vitro transcription. (B) RdRp assay using various in vitro synthesized RNA templates (indicated above the gel) in presence or absence of AmCPV RdRp. Sizes of the RNA markers (Ambion) are on the left. (C) Analysis of RNase A treated RdRp product under high (250 mM NaCl) and low salt (50 mM NaCl) conditions. (D) Effect of mutation in the GDD motif on RdRp activity of AmCPV S2 encoded protein using in vitro transcribed 3′ (−) strand of AmCPV S2 RNA as template. (E) Assay of TNTase activity in AmCPV S2 encoded protein.

Fig. 7

(A) Synthesis of product RNA using AmCPV RdRp using in vitro transcribed 500 nt long 3' positive strand AmCPV S2 RNA as template in respect of time. At the times indicated, aliquots were removed from the reaction mixture and the size of labeled product RNA was analyzed by formaldehyde agarose gel electrophoresis, and autoradiographed. (B) Plot showing the size of product RNA vs. reaction time. The straight line was obtained by linear regression.