Profiling the substrate specificity of viral protease VP4 by a FRET-based peptide library approach.

Knowing the substrate specificity of a protease is useful in determining its physiological substrates, developing robust assays, and designing specific inhibitors against the enzyme. In this work, we report the development of a combinatorial peptide library method for systematically profiling the substrate specificity of endopeptidases. A fluorescent donor (Edans) and quencher (Dabcyl) pair was added to the C- and N-termini of a support-bound peptide. Protease cleavage of the peptide removed the N-terminal quencher, resulting in fluorescent beads, which were isolated and individually sequenced by partial Edman degradation and mass spectrometry (PED-MS) to reveal the peptide sequence, as well as the site of proteolytic cleavage. The method was validated with bovine trypsin and Escherichia coli leader peptidase and subsequently applied to determine the substrate specificity of a viral protease, VP4, derived from the blotched snakehead virus (BSNV). The results show that VP4 cleaves peptides with a consensus sequence of (Abu/Ala/Pro)-X-Ala downward arrowX, in agreement with the previously observed cleavage sites in its protein substrates. Resynthesis and a solution-phase assay of several representative sequences against VP4 confirmed the library screening results.

Activation mechanism of a noncanonical RNA-dependent RNA polymerase.

Two lineages of viral RNA-dependent RNA polymerases (RDRPs) differing in the organization (canonical vs. noncanonical) of the palm subdomain have been identified. Phylogenetic analyses indicate that both lineages diverged at a very early stage of the evolution of the enzyme [Gorbalenya AE, Pringle FM, Zeddam JL, Luke BT, Cameron CE, Kalmakoff J, Hanzlik TN, Gordon KH, Ward VK (2002) J Mol Biol 324:47-62]. Here, we report the x-ray structure of a noncanonical birnaviral RDRP, named VP1, in its free form, bound to Mg(2+) ions, and bound to a peptide representing the polymerase-binding motif of the regulatory viral protein VP3. The structure of VP1 reveals that the noncanonical connectivity of the palm subdomain maintains the geometry of the catalytic residues found in canonical polymerases but results in a partial blocking of the active site cavity. The VP1-VP3 peptide complex shows a mode of polymerase activation in which VP3 binding promotes a conformational change that removes the steric blockade of the VP1 active site, facilitating the accommodation of the template and incoming nucleotides for catalysis. The striking structural similarities between birnavirus (dsRNA) and the positive-stranded RNA picornavirus and calicivirus RDRPs provide evidence supporting the existence of functional and evolutionary relationships between these two virus groups.

The structure of a birnavirus polymerase reveals a distinct active site topology.

Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 A structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.

Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus (BSNV).

Blotched snakehead virus (BSNV) is a member of the Birnaviridae family that requires a virally encoded protease known as VP4 in order to process its polyprotein into viral capsid protein precursors (pVP2 and VP3). VP4 belongs to a family of serine proteases that utilize a serine/lysine catalytic dyad mechanism. A mutant construct of VP4 with a short C-terminal truncation was overexpressed in Escherichia coli and purified to homogeneity for crystallization. Using the sitting-drop vapour-diffusion method at room temperature, protein crystals with two distinct morphologies were observed. Cubic crystals grown in PEG 2000 MME and magnesium acetate at pH 8.5 belong to space group I23, with unit-cell parameters a = b = c = 143.8 angstroms. Trigonal crystals grown in ammonium sulfate and glycerol at pH 8.5 belong to space group P321/P312, with unit-cell parameters a = b = 158.2, c = 126.4 angstroms.

Comparison of the RNA polymerase genes of marine birnavirus strains and other birnaviruses.

The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3-99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4-20.8% divergences in the coding region, which gave 10.1-11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1-85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1-95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III.

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage.

Template-dependent polynucleotide synthesis is catalyzed by enzymes whose core component includes a ubiquitous alphabeta palm subdomain comprising A, B and C sequence motifs crucial for catalysis. Due to its unique, universal conservation in all RNA viruses, the palm subdomain of RNA-dependent RNA polymerases (RdRps) is widely used for evolutionary and taxonomic inferences. We report here the results of elaborated computer-assisted analysis of newly sequenced replicases from Thosea asigna virus (TaV) and the closely related Euprosterna elaeasa virus (EeV), insect-specific ssRNA+ viruses, which revise a capsid-based classification of these viruses with tetraviruses, an Alphavirus-like family. The replicases of TaV and EeV do not have characteristic methyltransferase and helicase domains, and include a putative RdRp with a unique C-A-B motif arrangement in the palm subdomain that is also found in two dsRNA birnaviruses. This circular motif rearrangement is a result of migration of approximately 22 amino acid (aa) residues encompassing motif C between two internal positions, separated by approximately 110 aa, in a conserved region of approximately 550 aa. Protein modeling shows that the canonical palm subdomain architecture of poliovirus (ssRNA+) RdRp could accommodate the identified sequence permutation through changes in backbone connectivity of the major structural elements in three loop regions underlying the active site. This permutation transforms the ferredoxin-like beta1alphaAbeta2beta3alphaBbeta4 fold of the palm subdomain into the beta2beta3beta1alphaAalphaBbeta4 structure and brings beta-strands carrying two principal catalytic Asp residues into sequential proximity such that unique structural properties and, ultimately, unique functionality of the permuted RdRps may result. The permuted enzymes show unprecedented interclass sequence conservation between RdRps of true ssRNA+ and dsRNA viruses and form a minor, deeply separated cluster in the RdRp tree, implying that other, as yet unidentified, viruses may employ this type of RdRp. The structural diversification of the palm subdomain might be a major event in the evolution of template-dependent polynucleotide polymerases in the RNA-protein world.

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus.

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.