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. 2004 May;78(9):4397-407.
doi: 10.1128/jvi.78.9.4397-4407.2004.

Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus

Christopher T Cornell  1 , Rushika Perera, Jo Ellen Brunner, Bert L Semler
Affiliations

Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus

Christopher T Cornell et al. J Virol. 2004 May.

Abstract

The viral RNA-dependent RNA polymerase (3D(pol)) is highly conserved between the closely related enteroviruses poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3). In this study, we generated PV1/CVB3 chimeric polymerase sequences in the context of full-length poliovirus transcripts to determine the role of different subdomains within the RNA-dependent RNA polymerase of PV1 that are required for functions critical for RNA replication in vitro and in cell culture. The substitution of CVB3 sequences in the carboxy-terminal portion (thumb subdomain) of the polymerase resulted in transcripts incapable of RNA replication. In contrast, three of the seven chimeras were capable of synthesizing RNA, albeit to reduced levels compared to that of wild-type PV1 RNA. Interestingly, one of the replication-competent chimeras (CPP) displayed an inability to generate positive strands, indicating the presence of amino-terminal sequences within the 3D polymerase and/or the 3D domain of the 3CD precursor polypeptide that are necessary for the assembly of strand-specific RNA synthesis complexes. In some constructs, the partial reestablishment of PV1 amino acid sequences in this region was capable of rescuing RNA replication in vitro and in cell culture.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic of chimeric PV1/CVB3 transcripts. The top of the panel shows the organization of a PV1 cDNA containing a 5′ cis-acting hammerhead ribozyme. Transcriptions with T7 RNA polymerase result in the production of wild-type or chimeric PV1/CVB3 transcripts with precise 5′ ends. Shown below the parent transcript are the different RNAs used in the present study containing either a wild-type or chimeric PV1/CVB3 polymerase domain. Portions of the polymerase gene containing CVB3 sequence are indicated by the hatched boxes, along with the amino acid numbers corresponding to the PV1/CVB3 sequence junctions in each construct. (B) Schematic indicating which secondary structure(s) within the poliovirus 3D polymerase are affected by CVB3 amino acid substitutions generated in panel A, along with “fingers,” “thumb,” and “palm” subdomain designations. §, based on previously published data (9).
FIG. 2.
FIG. 2.
In vitro translation and RNA replication of wild-type and chimeric PV1/CVB3 transcripts. (A) α-32P-labeled RNA replication reactions were incubated with 1.3 μg of virion RNA, wild-type RzPV1 transcript, or chimeric RzPV1 transcript RNA and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes 1 and 2 are positive controls utilizing purified PV1 virion RNA (vRNA) and wild-type RzPV1 transcript, respectively. Lane 3 is a negative control in which the wild-type RzPV1 replication reaction was supplemented with 2 mM guanidine hydrochloride (GuHCl) to inhibit RNA synthesis. The identity of the full-length RzPV1 transcript RNA utilized in each reaction is indicated above each lane. The mobilities (visualized by ethidium bromide staining) of single-stranded virion RNA (ssRNA) and the 28S and 18S rRNAs are indicated to the right of the panel. Also shown is the predicted mobility of the replicative intermediate/replicative form (RI/RF), corresponding, in part, to negative-strand synthesis. The autoradiogram shown below is a longer exposure of the same gel. (B) SDS-polyacrylamide gel analysis of [35S]methionine-labeled translations from the corresponding reactions shown in panel A. The identities of the precursor and mature cleavage products generated during translation and processing in vitro are indicated on the left side of the panel. Note the different mobilities of the chimeric 3CD and 3D polypeptides in each lane due to the substitution of PV1 amino acids with those from CVB3.
FIG. 3.
FIG. 3.
(A) Sequence alignment of the amino-terminal one-third of PV1 and CVB3 3D polymerases, which share ca. 75% amino acid identity. Shaded white letters show regions of sequence divergence, and the amino acids in the top of the thumb and base of the fingers are boxed (aa 12 to 37 and 67 to 97). In the alignment, circled letters A to D indicate junctions that are the boundaries for regions 1, 2, and 3 (i.e., region 1 consists of CVB3 amino acids from junction A to B, region 2 consists of amino acids from junction B to C, etc.). (For the PV1 sequence, see reference ; for the CVB3 sequence, see reference 19). (B) Schematic of amino-terminal PV1/CVB3 chimeric luciferase replicons. The plasmid construct (top of panel) pRib(+)RLucM contains a cis-acting 5′ hammerhead ribozyme and the firefly luciferase gene in place of the viral capsid sequence. In vitro transcriptions with T7 RNA polymerase yield the RNAs appearing below, with different regions (shown by hatched boxes) of the amino-terminal one-third of the PV1 polymerase changed to CVB3 sequence. Also shown are region number designations used in the nomenclature for these constructs. The right portion of the panel indicates the location of the chimeric substitutions based on the published three-dimensional structure of the PV1 3D RNA polymerase, although most substitutions (other than αA within the base of the fingers) are in an unresolved region of the structure. †, based on previously published data (9).
FIG. 4.
FIG. 4.
Luciferase replicon kinetics. Replicon transcripts shown in Fig. 3B were transfected into HeLa monolayers as described in Materials and Methods. At 2, 4, 6, 8, 10, and 14 h posttransfection, cells were harvested and luciferase activity measured. The y axis shows log10 units of luciferase activity, and the x axis shows time (in hours) posttransfection. Luciferase units in the presence (•) and absence (▪) of guanidine-HCl (an inhibitor of RNA replication) are shown in each graph. Although not readily visible, standard deviations (as error bars) are given for each data point.
FIG. 5.
FIG. 5.
In vitro translation and RNA replication of wild-type (full-length and luciferase replicon) and amino-terminal chimeric PV1/CVB3 luciferase replicon transcripts. (A) RNA replication carried out as described in the legend for Fig. 2A, utilizing full-length PV1 transcript (lane 1) and wild-type (lane 2) or amino-terminal chimeric luciferase replicon RNA (lanes 4 to 10). The autoradiogram shown below is a longer exposure of the same agarose gel. (B) [35S]methionine-labeled translations from the corresponding reactions shown in panel A, carried out as in Fig. 2B. The mobility of firefly luciferase (observed in lanes 2 to 10) is indicated by an asterisk to the right of the panel.
FIG. 6.
FIG. 6.
Molecular modeling of 3D polymerase interfaces. The coordinates of the three-dimensional structure of the PV1 3D polymerase (PDB identification number 1RDR) (9) were used to construct models of selected PV1/CVB3 chimeric 3D polymerase molecules by using the program O (14). Residues unique to CVB3 3D were substituted for those of PV1 3D and the resulting effects on intra- and intermolecular interactions were analyzed (9). (A to C) Four polymerase molecules (colored blue and green), arranged as described in the published three-dimensional structure, with the proposed interfaces I and II (boxes X and Y, respectively) depicted and circled letters highlighting subdomains within the 3D polymerase (T, thumb; P, palm; F, fingers). The amino acid side chains changed to CVB3 sequence in chimera CPP (A), PCP (B), and PPC (C) are shown in red. Boxes X and Y indicate the regions shown in panels E and D, respectively. (D) Ribbon diagram of interface II. One polymerase is shown in green and the adjacent polymerase molecule is shown in blue, with the amino acids in each that participate in the formation of proposed interface II colored yellow and aqua, respectively. The amino acid side chains (shown in red) changed to CVB3 sequence in the CPP chimera are labeled. Interface II is indicated by the dashed line, and the amino acids comprising the two faces of proposed interface II (aa 12 to 37 [top of thumb] and 67 to 97 [base of fingers]) are numbered. (E) Ribbon diagram of interface I, with the same coloring scheme as in panel D, with labeled side chains (shown in red) representing amino acids changed to CVB3 in the PPC chimera. The locations of α helices I, L, and N are depicted. Interface I is indicated by a dashed line.
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