Identification And Characterization Of Flock House Virus Protein A RNA-dependent RNA Polymerase Activities

Flock House virus (FHV) was originally isolated from grass grubs collected in the vicinity of the former Flock House agricultural station on the North Island of New Zealand. FHV is a nonenveloped, icosahedral virus belonging to the family Nodaviridae, whose members are characterized by genomes consisting of two molecules of single-stranded, positive-sense RNA. FHV has furthermore been classified into the genus Alphanodavirus, whose members naturally infect insects, as opposed to the genus Betanodavirus, whose members are pathogenic to fish.The combined length of RNA1and RNA2is4507nucleotides, one of the smallest genomes of all known animal viruses. This property, together with the simple genomic organization of FHV and its abundant replication in a wide variety of cells, makes FHV a highly tractable system for studies on a variety of basic aspects of virology, including RNA replication, specific genome packaging, and virus structure and assembly.RNA1contains an open reading frame for the synthesis of protein A (112kD), which serves as the RNA-dependent RNA polymerase (RdRp) for the amplification of both genomic strands as well as a387nucleotide subgenomic RNA (RNA3). A domain spanning amino acids513-752of protein A is most probably central to its RNA replication activities. This domain contains six of the eight conserved motifs previously identified for RdRps of (+)-strand RNA viruses. One of these motifs, a glycine-aspartateaspartate (GDD) box that is required for the polymerization activities of all known polymerases, is also required for protein-A-dependent RNA replication.In this study, we expressed and purified FHV protein A as an N-terminal maltoses-binding protein (MBP) tagged protein (MBP-ProtA) in E. coli. Moreover,, we conducted mutations in the conversed motifs GDD. Using the plus-strand RNA13’ UTR (+)1-191and minus-strand RNA13’ UTR (-)1-201as templates, we perforemed Northern blot and RT-PCR to study the mechanism of initiating FHV RNA1sythesis by protein A. The results showed that FHV protein A preferred to initiate either plus-or minus-strand viral RNA synthesis by primer-independent de novo manner but not primer-dependent RNA sythesis in vitro.Using (-)1-201as template, the optimal reaction conditions were found——30℃, pH8.0,1.0mM Mn2+, only Mn2+could support de novo initiation, Mg2+couldn’t.We uncovered that FHV protein A can add Digoxingenin-11-UTP to the3’end of template RNAs.Furthermore, we evaluated the effects of3’nucleotide deletions of (+)1-191on RdRP-catalyzed RNA synthesis. RNA synthesis initiation could tolerate the loss of two nucleotides in the3’end, whereas more nucleotide loss,about13nucleotides, would inhibit the RNA synthesis in a great degree. When the49nucleotides in the3’end of the (+)1-191, the RNA products was little, almost can not be detected. Also, we evaluated the effects of3’nucleotide deletions of (-)1-201on the RDRP-catalyzed positive strand synthesis. When one or two nucleotides were lost at the3’end of the (-)1-201substrate, the RDRP-catalyzed RNA synthesis could be detected, whereas more nucleotide loss completely blocked the RDRP reaction. RNA synthesis was more decreased for (-)A3U-201than for (-)A3G、(-)A3C、(-)A3U、(-)A3G-201、(-)A3C-201.We also evaluated the effects of3’nucleotide deletions of (-)1-201on the TNTase-catalyzed nucleotide addition. When one, two or three nucleotides were lost at the3’ end of the (-)1-201substrate, the TNTase-catalyzed nucleotide addition could be detected, even more higher efficiency than the (-)1-201as the template, whereas four nucleotides loss would block the most the TNTase reaction and more nucleotide loss would completely block the TNTase reactionAt last,we found terminal nucleotidyl transferase (TNTase) activity of protein A functions could restore some3’-proximal nucleotides of template RNA as a potential mechanism for rescuing3’-terminal nucleotide loss. When protein A was supplemented in the TNTase reaction, the reacted (-)RNA1templates(-)4-201and (-)A3U-201, can be efficiently replicated in the RdRP reaction. These data indicate that the TNTase activity of protein A can function to restore the replicability of the RNA1template by repairing the3’-terminal nucleotide loss.