The rotavirus polymerase domain resembles closely that of other viral RdRPs, and its N- and C-terminal domains are very similar to those of reovirus λ3. Moreover, both the conformation of the bound template and the way in which the templating bases are wedged against a segment of the fingers domain are conserved among all polymerases with a right-handed core for which the structure of a catalytic complex is known. Sequence-specific recognition of an RNA template sequence has not previously been documented, however, and the binding of template in an overshot register is also unusual. These similarities and differences allow us to correlate structural features with various aspects of VP1 activity, both in its enzymatic mechanism and in its broader biological function.
A model summarizing events in dsRNA synthesis by VP1 can be developed from comparisons of our crystallographic results with those obtained earlier for λ3 initiation and elongation complexes (). The rotavirus polymerase specifically recognizes its +RNA template by forming hydrogen bonds with the bases of the 3′CS+ UGUG residues. These interactions, combined with additional contacts to the sugar-phosphate backbone, anchor the 3′CS+ such that its C1 residue lies one nucleotide beyond the initiation register. This recognition complex is catalytically inactive. During assembly of progeny cores, VP1/+RNA complexes interact with VP2, inducing conformational changes that lead to initiation. The expected changes include a shift in the priming loop from a retracted to an extended position, allowing the initiating nucleotide to be stabilized in the P site. Nucleotides at the P and N positions may help bring C1 and C2 into alignment for initiation, and conformational changes elsewhere in the molecule must also have a role, as the priming loop does not communicate directly with the molecular surface. Correct alignment and base pairing will lead to formation of the first phosphodiester bond of the −RNA product. Once the initiating dinucleotide has formed, the extended priming loop will impede transfer of the RNA product. Retraction of the priming loop has been seen in λ3 as the polymerase transitions from initiation to elongation mode (Tao et al., 2002
). In λ3, the elongating dsRNA product can move through the −RNA/dsRNA exit tunnel without impediment. In VP1, the C-terminal plug must be displaced from this exit tunnel. Forward translocation of the dsRNA product, driven by nucleotide hydroylsis, may be sufficient to open the channel, as the His-tagged plug in our protein preparations, which bind Ni-NTA, appears to emerge spontaneously. The plug does not fill the tunnel, and there is enough space to accommodate a bypassing single-stranded RNA. If it remained in the −RNA/dsRNA exit tunnel during transcription, the plug could have a regulatory role in the switch from replication to transcription.
Proposed model of RNA synthesis by VP1
Following synthesis, the dsRNA products of replication remain in cores, where they act as templates for multiple rounds of transcription. The 3′CS- (AAAAGCC-3′) of the −RNA template, although anchored in the template entry tunnel in our crystalline complex, does not contact VP1 in a sequence-specific manner. Thus, the rotavirus polymerase specifically recognizes only the 3′CS+ of the +RNA template for packaging and dsRNA synthesis. Highly specific screening of the −RNA templates for transcription may not be needed, as they are confined to the interior space of the core. Moreover, retention of the capped 5′ end of the +RNA in the cap-binding site will position the 3′ end of the −RNA near the template entry channel and favor its selective insertion.
All viruses of the many Reoviridae
genera have similarly structured cores (see, for example, Lawton et al, 2000
), with a tightly packed shell composed of 60 asymmetric dimers of VP2 or a homologous core shell protein (e.g.
, λ1 in orthoreoviruses and VP3 in orbiviruses). The shell is an icosahedrally symmetric array of 12 core shell protein decamers, each centered on a fivefold axis and each associated with a single (internal) polymerase molecule. The structures described in this paper and the properties of the viral cores suggest a simple mechanism for specific, +RNA packaging in a rotavirus infection. (1) Recognition of the 3′CS+ establishes stable, but inactive, VP1/+RNA complexes. (2) Association of this initial complex with a decamer of VP2 (or VP2 homolog) creates the basic assembly unit of the core. In all Reoviridae
family members, one or more copies of an enzyme required for capping the transcript (e.g.
, VP3 of rotaviruses) must also be recruited. Association of the polymerase with the core shell protein subunits could also precede RNA recognition. (3) Twelve such basic assembly units (e.g.
for rotaviruses) come together to form a functional core. (4) Additional proteins (e.g., VP6, which forms the second “layer” of the rotavirus DLP) then stabilize the core and mediate interaction with outer-shell components (e.g.
, rotavirus VP4 and VP7). Recombinant polymerases from other Reoviridae
family members (e.g.
, reovirus and bluetongue virus) have modest activity in vitro
in the absence of the core shell protein. Whether the purified forms of the reovirus and bluetongue virus polymerases represent the structural and functional equivalents of the VP1 apoenzyme, or possibly a later activated form, remains to be determined.