Processing of the 3C/D Region of the Deformed Wing Virus (DWV)

Abstract

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1′ G2119 (KPQ/GST) as well as P1 Q2393 and P1′ S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

Characterization of a Molecular Clone of Deformed Wing Virus B

“…DWV-A was quantified as described earlier [ 36 ]. To measure and compare viral RNA replication products against remnants of in vitro-transcribed RNA, bee pupae received 1 µg of RNA from the rDWV-B strain Austria-SB22 and the rDWV-A strain 1414, as well as from two non-replicative control genomes, namely, rDWV-B-E 1102 Stop and rDWV-A-Q 2118 A [ 19 ]. Further controls encompassed the field virus (wtDWV-B, SB22) and mock-transfected bee pupae.…”

“…Antibody binding was detected utilizing chemiluminescence (ECL-Prime; GE Healthcare; Chicago, IL, USA) and digital imaging (Odyssey M; Li-Cor; Lincoln, NE, USA). Mab VP1A1 against the VP1 of DWV [ 36 ], Mab 55A10 anti-Pol [ 19 ], and Mab B-2 anti-GFP (sc-9996; Santa Cruz; Dallas, TX, USA) were employed as specified.…”

“…The typical replication enzymes in the P2 and P3 cassettes, such as helicase (2C-like), protease (3C-like), and RNA-dependent RNA polymerase (3D-like), have already been identified by Lanzi et al, along with their potential processing sites [ 15 ]. Only recently, Yuan et al (N-terminus of 3C-like protease) and our group (N-terminus 3C-like protease and N-terminus 3D-like polymerase) uncovered the precise processing of the 3CL/3DL region [ 18 , 19 ].…”