Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites

Abstract

The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine-lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)-X-Ala Ala motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis-and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease.

Genome Assembly and Particle Maturation of the Birnavirus Infectious Pancreatic Necrosis Virus

“…3D). By mutagenesis of recombinant proteins, the cleavage sites of birnavirus pVP2 (38,56) were identified and some of them were later shown to be essential for viral viability, indicating that maturation of IBDV pVP2 is also critical for infectivity (12). On the other hand, the viral polyprotein is processed and cleaved at the pVP2-VP4 and VP4-VP3 junctions by the viral protease VP4 (1,45,46,56), which is a Ser-Lys protease (56) and shares homology to bacterial Lon proteases (3).…”

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

“…Segment A (3.3 kb) has two partially overlapping open reading frames. Open reading frame A-1 encodes a polyprotein that is autocatalytically cleaved to yield the immature outer capsid protein pVP2, the viral protease VP4 and the ribonucleoprotein VP3 (Sánchez & Rodriguez, 1999;Lejal et al, 2000;Da Costa et al, 2002;Luque et al, 2009). The pVP2 protein is further processed to yield the mature VP2, the immunodominant antigen of IBDV (Fahey et al, 1991).…”

Blotched Snakehead Virus Is a New Aquatic Birnavirus That Is Slightly More Related to Avibirnavirus Than to Aquabirnavirus

“…The polyprotein is cotranslationally processed through the proteolytic activity of VP4 to generate pVP2, VP4, and VP3. Cleavage sites at pVP2-VP4 and VP4-VP3 junctions have been identified for IPNV, IBDV, and DXV (11,26,32,33). For IBDV, the processing of pVP2 (amino acids [aa] 1 to 512) generates VP2 and four small peptides (aa 442 to 487 [M. Skinner, personal communication], 488 to 494, 495 to 501, and 502 to 512) (12).…”

“…This maturation cleavage process of pVP2 requires assembly of viral capsids (8). The IBDV and IPNV VP4 proteases have been shown to use a serine-lysine catalytic dyad to control the processing of the polyprotein (2,26,32).…”