Mutation Analysis of the GDD Sequence Motif of a Calicivirus RNA-Dependent RNA Polymerase

Abstract

The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.Rabbit hemorrhagic disease virus (RHDV) has been characterized as a member of the Caliciviridae (19, 21) and recently designated as the type species of the new genus Lagovirus (24). The viral genome is a positive polarity, single-stranded, polyadenylated RNA with approximately 7.4 kb, which has a virusencoded VPg protein covalently attached to its 5Ј end (15,16,28). Viral particles also encapsidate an abundant VPg-linked polyadenylated subgenomic RNA of about 2.2 kb (16). The data obtained from the in vitro translation (28), Escherichia coli expression studies (14), and detection of viral proteins after infection of cultured hepatocytes with RHDV (8) revealed that the viral RNA is translated into a polyprotein that is subsequently cleaved to give rise to mature structural and nonstructural proteins.The extensive sequence similarities between the RNA-dependent RNA polymerase (RdRp) 3D of picornavirus and the RHDV polyprotein cleavage product p58 (8,15,28) suggested that this polypeptide could have a similar role in RHDV genome replication. Studies from our laboratory have reported the successful expression of enzymatically active RHDV RNA polymerase (3D pol ) in E. coli (13). The recombinant polypeptide exhibited rifampin-and actinomycin D-resistant poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity by using synthetic RHDV subgenomic RNA as a template in the presence or absence of an oligo(U) primer (13).The availability of a growing number of complete sequences from the genomes of positive, negative, and double-stranded RNA viruses of plant and animal origin has enabled many groups to carry out sequence alignments of the RdRps in an attempt to identify regions essential for polymerase function, which should appear as most conserved (1,7,23). Currently, eight conserved motifs have been described (23), four of which (A, B, C, and D) are now known to be located in the catalytic portion of the "palm" domain (4) of all classes of polymerases. Motif C, which formed a "␤-strand, turn, ␤-strand" structure, contained a highly conserved GDD sequence found in all RdRps (7). This structure is very similar in all classes of polymerases and positions the two aspartate residues close to the conserved aspartate of motif A (4). The location ...

Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

“…Sapovirus 3D pol activity was found to depend on the presence of Mg 2ϩ or Mn 2ϩ , with a clear preference for Mn 2ϩ . This flexibility for metal ion usage has already been described for the 3D pol of norovirus (4, 17, 50, 51) but also for various viral RNA-dependent RNA polymerases (11,59), i.e., poliovirus and brome mosaic virus, where the RNA polymerase displays a preference for Mn 2ϩ as well (25). Our data also indicate that sapovirus 3D pol synthesizes RNA from heteropolymeric subgenomic RNA by a de novo initiation mechanism rather than by back priming.…”

“…This limitation in the cultivation of calicivirus has hampered studies on the replication strategies of these important human and animal pathogens. An alternative approach to cell-based studies has relied on structural and functional characterization in vitro of the viral enzymes putatively implicated in replication, such as the viral polymerase (4,17,21,43,44,50,51,59,60,62), the viral protease (5,40,45,(54)(55)(56)65), or the viral NTPase (36,48). Furthermore, reverse genetic systems for Norwalk virus (norovirus) (3,28) and porcine enteric calcivirus (sapovirus) have been developed (7,8).…”

Characterization of an Enteropathogenic Bovine Calicivirus Representing a Potentially New Calicivirus Genus

“…Identifying defined and conserved calicivirus structural-functional motifs and proteolytic cleavage sites predicted seven potential cleavage products of NB virus ORF-1. Their predicted order, NH 2 -P34-P37(2C)-P30-P7.6(VPg)-P20(3C)-P53(3D)-P57(VP1)-COOH, and projected functions (ATPase, VPg, 3C protease, RdRp, and capsid) confirm the NB virus as a member of the family Caliciviridae (8,13,55,64,74,79,80). An unusual feature of the predicted polypeptide of the 3C protease of the NB virus was the substitution of tyrosine (Y) for an aspartic acid (D) residue (underlined) in the characteristic GDCG motif that is found in all other caliciviruses.…”

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

“…Two Mn 2ϩ ions also coordinate to three highly conserved Asp residues and the NTP triphosphate moiety to mediate catalysis through the two-metal-ion mechanism (10,28) (Fig. 3).…”