Structural studies on Norwalk virus capsids

Norwalk virus, a member of the Caliciviridae, is a prototype human calicivirus that causes epidemic acute gastroenteritis (Kapikian et al 1996). Human caliciviruses have been very difficult to adapt to cell culture systems. The lack of a cell culture system or a practical animal model has to some extent limited the progress in understanding the molecular characteristics of the virus and its replication strategies. However, the successful cloning of the Norwalk virus genome and its expression using the baculovirus system has alleviated this problem and begun to provide a better insight into the epidemiological, immunological, biochemical and other functional properties of the virus (Jiang et al 1990, 1993, Estes & Hardy 1995, Clarke & Lambden 2001 this volume).

In contrast to rotaviruses, Norwalk virus and the other members of the Caliciviridae are significantly smaller (diameter ~ 400 A) and architecturally simpler viruses. The icosahedral capsids of caliciviruses are made of a single structural protein. In these viruses, 180 molecules of the structural protein form a T = 3 icosahedral capsid (Prasad et al 1994a,b) that contains the viral genome of positive sense single-stranded RNA of approximately 7.5 kb.

Atomic resolution structure of the "Norwalk virus capsid

Recently the X-ray crystallographic structure of the recombinant Norwalk virus capsid (Fig. 3) has been determined to 3.4 A resolution using a low-resolution cryo-EM structure as an initial phasing model (Prasad et al 1999). The structure of the capsid protein exhibits both classical and novel features. The N-terminal 220 residues constitute the S-domain and fold into a classical eight-stranded ^-sandwich. The rest of the sequence constitutes the protruding (P) domain and has a fold unlike any other viral protein. The P domain consists of two subdomains: P2 and P1 (Fig. 3b). An interesting discovery is that the polypeptide fold of the distal P2 domain is similar to that seen in the domain 2 of the EF-Tu protein, an important factor in the biosynthesis of proteins. The functional significance of this structural similarity is still unclear. The distal P2 domain, which exhibits large sequence variation between various Norwalk-like viruses, unlike the S and the P1 domains that are better conserved, may serve as a replaceable module to provide strain specificity. As observed in other T= 3 viruses (Rossmann & Johnson 1989), there are two distinct conformational dimeric states of the capsid protein: the 'flat' C/C dimers at the icosahedral twofold axes, and the 'bent' A/B dimers at the quasi twofold axes. During the assembly process, the dimer has to switch from one type to the other. Where is

FIG. 3. Structure of the Norwalk virus capsid. (a) Structure of the recombinant Norwalk virus capsid at 3.4 A resolution. (b) The ribbon representation of the capsid protein (C subunit) structure. The structure of the capsid protein is shown oriented such that the top is at the exterior of the capsid and the bottom faces the interior. Various domains and subdomains are indicated (Prasad et al 1999).

FIG. 3. Structure of the Norwalk virus capsid. (a) Structure of the recombinant Norwalk virus capsid at 3.4 A resolution. (b) The ribbon representation of the capsid protein (C subunit) structure. The structure of the capsid protein is shown oriented such that the top is at the exterior of the capsid and the bottom faces the interior. Various domains and subdomains are indicated (Prasad et al 1999).

the switch? We have seen that the N-terminal arm of the B subunit is more ordered than that in the A or C subunits. Based on our structural observations, we have proposed that: (1) the N-terminal 20 residues of the capsid protein may serve as a switch for the capsid assembly, and (2) the hinge region together with the C-terminal residues in the P1 domain that forms hydrogen bonds with the S domain residues are important for conferring the appropriate curvature for the dimers to assemble onto a T = 3 icosahedral shell. This atomic resolution model can now allow a systematic mutational analysis, which is in progress, in order to understand the assembly mechanism and other functional properties of Norwalk virus and other caliciviruses.

Our work is supported in part by grants from the NIH (AI36040 and DK 31044, AI46581), and the R. W. Welch Foundation.

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