Caliciviridae represent a family of small positive-strand RNA viruses. They comprise four genera (
22) distinguished by their host range and genome organization (
12,
13). Two genera, the Sapporo-like viruses and the Norwalk-like viruses (NLVs), are highly contagious human pathogens that are responsible for outbreaks of epidemic acute gastroenteritis (
12). NLVs, also called small round structured viruses, are currently divided into two genogroups based on nucleotide and amino acid sequence diversity (
2,
23,
37,
64). The prototype Norwalk virus and Southampton virus (SHV) belong to genogroup 1. At present, there is neither a cell culture nor an animal system available to study the replication of NLVs.
Sequence analyses of the ORF 1 of caliciviruses have revealed the presence of motifs in the primary translation product that are associated with distinct functions of nonstructural proteins encoded by picornaviruses and other plus-strand RNA viruses (
12). These functions include a trypsin-like cysteine proteinase, an RNA-dependent RNA polymerase (RdRp), and a putative superfamily 3 (SF3) helicase (
21). Most attention has been focused on the proteinase, which has been shown to be related to picornavirus 3C
pro based on similarity of sequence and function (
12). The RdRp of rabbit hemorrhagic disease virus has been shown to synthesize RNA in a primer- and template-dependent manner (
40) as has been demonstrated earlier for picornavirus 3D
pol (
19). No function has yet been demonstrated for a calicivirus-encoded putative SF3 helicase. The observation that the translation product of calicivirus ORF 1 shares sequence motifs with picornavirus nonstructural proteins may indicate an evolutionary relationship between the two virus families (
23,
24) and similar genome replication strategies.
Picornaviruses have been extensively studied, and among them poliovirus (PV) is one of the best characterized (
66). The nonstructural protein 2C contains the motifs A, B, and C related to nucleoside triphosphatase (NTPase) and possibly helicase activity (
38). Generally, motifs A and B, first described by Walker et al., appear in a variety of NTP-binding proteins of various functions (
63). Motif C consists of an invariant asparagine residue located at a distinct distance downstream of motif B (
21). Motif C is distinctive for SF3 helicases encoded by small DNA and RNA viruses (
21). Protein 2C of PV and Echovirus 9, both members of the genus
Enterovirus (EV), have been demonstrated to exhibit NTPase activities (
32,
41,
52,
55). Recently, we have shown that a bacterially expressed fusion protein of glutathione
S-transferase (GST) and PV 2C hydrolyzed ATP at least 70-fold more efficiently than other NTPs (
52). Mutations in motif A, B, or C abolished ATPase activity. The ATPase activity was strongly inhibited by 1 mM guanidine hydrochloride (
52), which at similar concentrations inhibits the RNA replication of PV and other picornaviruses (
4,
43,
53,
66). Mutations in PV 2C that confer a guanidine-resistant or -dependent virus phenotype covaried with increased guanidine tolerance of the ATPase (
52). On the basis of these data, we refer conveniently to this protein as 2C
ATPase (
52).
SHV was isolated originally from a stool sample of a 2-year-old child during a family outbreak of acute gastroenteritis in Southampton, United Kingdom (
36). Lambden et al. have constructed a full-length cDNA of the SHV genome and elucidated the sequence (
36). In vitro translation of ORF 1 combined with site-directed mutagenesis and immunodetection of the processing products revealed that the 3C-like proteinase releases a protein of 41 kDa (p41) that contains motifs A to C (
38). Using bacterially expressed and purified p41 of SHV we show for the first time an NTP-binding and -hydrolyzing activity associated with a calicivirus protein. The NTPase activity was independent of single-stranded nucleic acids. Comparison of calicivirus-encoded proteins with their picornavirus counterparts offers an additional opportunity to learn more about the molecular biology of human caliciviruses, a group of agents that are serious human pathogens (
44).
DISCUSSION
NLVs are human enteric caliciviruses for which no cell culture or small-animal system is available. Consequently, the tools for the study of virus replication and mechanisms of viral pathogenicity are restricted to in vitro assays. These assays have mostly addressed the proteolytic activity of the virally encoded trypsin-like cysteine proteinase (
12), an enzyme responsible for the cleavage of the ORF 1-encoded polyprotein. One of the cleavage products is p41, a protein with sequence motifs that suggest a relationship to SF3 helicases. In this study, we have addressed NTP binding, NTP hydrolysis, and helicase activities of SHV p41. By sequence comparison, we found regions of high similarity between NLV p41 and EV 2C.
Using a bacterially expressed GST-p41 fusion protein, we demonstrated that p41 bound ATP in a manner that required an intact motif A. P41 was able to hydrolyze ATP at a rate of up to 1.4 s
−1, which is slightly higher than that reported for PV 2C
ATPase (0.9 s
−1) (
52). In contrast to PV 2C
ATPase, which prefers strongly ATP as substrate (
52), p41 was found to be a promiscuous NTPase with a marginal preference for ATP over GTP and then CTP/UTP. To our surprise, the ATPase activity of p41 was most efficient at 15°C and in the presence of relatively high (≥3.5 mM) concentration of manganese ions. Optimal enzyme activity under rather nonbiological conditions may be explained by missing co factors that possibly play a role in the infected cell.
The NTPase activity of SHV p41
NTPase was neither dependent on nor stimulated by homopolymeric RNA. A stimulation of NTPase activity by RNA is a common property of helicases (
29). Moreover, whereas an RNA-DNA heteroduplex was readily unwound by HCV NS3, an RNA helicase (
30), this heteroduplex was not unwound by p41
NTPase. It is noteworthy that numerous attempts in several laboratories (
52,
55; H. Shimizu, personal communication) have also failed to identify a helicase activity for picornavirus protein 2C. Thus, although 2C-like proteins like p41 of caliciviruses and 2C of picornaviruses have adopted the NTP-binding and hydrolysis motifs of SF3 helicases, they seem to use their ability to hydrolyze NTPs for a function distinct from nucleic acid unwinding. Under optimal assay conditions, the ATPase activity of p41
NTPasewas sensitive to the presence of homopolymeric RNA, an observation arguing against helicase activity and suggesting that the NTPase activity may need to be down regulated at some point during viral replication.
The NTPase activity of p41
NTPase is the third enzymatic function identified for a calicivirus-encoded protein. In contrast to the proteolytic and RNA-synthesizing activities, the significance of the NTPase, however, is not obvious. In a reconstituted in vitro system, PV RNA polymerase (3D
pol) is able to initiate RNA synthesis in the absence of 2C
ATPase (
47). However, in an in vitro translation-transcription system primed with PV RNA, initiation of RNA synthesis required the guanidine-sensitive function (
4,
43), which has been identified to be the ATPase activity of 2C
ATPase (
52). 3D
pol has also been demonstrated to possess an unwinding activity (
10), allowing the polymerase to melt RNA duplexes of more than 1,000 bp in length while elongating a nascent RNA chain. However, mutations in motifs characteristic for SF3 helicases in 2C
ATPasedebilitate PV RNA replication in the infected cell (
42,
61). Thus, it seems that functions of 2C-like proteins are indispensable in the infected cell but dispensable in some biochemical reactions involving highly purified components such as initiation of RNA synthesis (
47) and RNA duplex unwinding (
10).
A large body of genetic and biochemical information has suggested that protein 2C of picornaviruses is involved in many processes during virus replication, yet its precise mechanism of function has not been determined. Inhibitors such as guanidine hydrochloride, benzimidazole derivatives, and derivatives of hydantoin target functions of 2C required for RNA replication (
4,
43,
66) and encapsidation (
62). Expression of picornavirus protein 2C in mammalian cells results in the formation of vesicles (
1,
11,
59,
60). In infected cells, these virus-induced vesicles are tightly associated with the viral replication complexes (
7,
9), the sites of RNA replication and virus assembly (
6,
50). Protein 2C, as well as the processing precursor 2BC, are believed to spatially organize the replication complex (
6,
8). Such a role of protein 2C is supported by its membrane (
18,
60) and RNA-binding (
3,
54) properties.
The morphology of the novel membranous structures that appear in eukaryotic cells expressing protein 2C of PV or hepatitis A virus has been studied in detail by electron microscopy (
1,
11,
59,
60). A mutation in motif A of PV 2C
ATPaseresulted in membrane structures distinct from those induced by
wt 2C (
11). The mutation corresponds to our mutation K168Q in p41
NTPase, which affected ATP binding (Fig.
2B). Thus, NTP-bound 2C and p41 may have a structural function that is regulated by the intrinsic NTPase activity. One may speculate that 2C-like proteins are involved in RNA replication and encapsidation by creating a dynamic structural scaffold that responds to the changing requirements during virus replication. Experiments that address the biochemical functions of SHV p41
NTPase and their effects on eukaryotic cells might help to clarify the mechanism by which caliciviruses and picornaviruses replicate and cause disease.
Caliciviridae and
Picornaviridae are evolutionary only distantly related (
5,
24). Both families include numerous pathogens that infect a wide array of different tissues in a variety of hosts. It is thus not surprising that 2C-like proteins have diverged to accommodate different protein-protein and/or protein-RNA interactions occurring during replication in a tissue-and host-specific environment (
24). To keep sequence diversity minimal, we restricted our sequence comparison to the 2C-like proteins of NLVs and EVs. Both genera include predominantly human pathogens, and both contain viruses that infect the gastrointestinal tract. Comparing the consensus sequences obtained from each genus, regions of high similarity have been identified. These regions precede the known motifs A to C by distinct spacing and were thus termed A′ to C′. A′ to C′ overlap with the more extensive sequence homologies in these regions found in alignments of picornavirus sequences (
67). In addition, two invariant arginine residues, called motif D, were found at a constant distance downstream of motif C. Motif D has been found in other picorna- and caliciviruses (
28,
67) but is not present in the SF3 helicases for which helicase activity has been demonstrated, such as simian virus 40 large T antigen and E1 of papillomaviruses (not shown). It will be interesting to see whether motifs A′, B′, C′, and D are involved in unique functions of 2C-like members of SF3 helicases.
Individual secondary structure predictions for NLV p41 and EV 2C revealed that the seven conserved motifs map to a central domain consisting of almost identical patterns of α helices and β sheets. The same result was obtained when 2C-like protein sequences of picornaviruses, caliciviruses, and picorna-like plant viruses were all aligned and used as input for a structure prediction (
60). Both approaches combined with sequence comparisons support a model in which the central α/β domain carries out the core function of 2C-like proteins (
60). Taken together, our results suggest that SHV p41 and most likely the corresponding polypeptides of all caliciviruses are NTPases that are functionally related to protein 2C of picornaviruses.