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. 1999 Jan;73(1):427-35.
doi: 10.1128/JVI.73.1.427-435.1999.

Functional coupling between replication and packaging of poliovirus replicon RNA

C I Nugent  1 , K L Johnson, P Sarnow, K Kirkegaard
Affiliations

Functional coupling between replication and packaging of poliovirus replicon RNA

C I Nugent et al. J Virol. 1999 Jan.

Abstract

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

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Figures

FIG. 1
FIG. 1
Experimental design of trans-encapsidation assays. (A) RNA molecules used for infections, transfections, and RNase protection experiments are diagrammed. Wild-type poliovirus RNA, with the coding regions of the individual proteins within the polyprotein, is compared to the genome of 3NC-202guaR virus. Two point mutations in the coding region for 2C (24) and an 8-nt insertion in the 3′ noncoding region (50) of 3NC-202guaR are indicated. R2Z and R3Z RNAs contain deletions of nt 1215 to 2996 and 1215 to 2510, respectively (22). Both R2Z and R3Z also have 68 nt of heterologous sequence inserted into their 5′ noncoding region (53). RNA probes of 203 nt, used to detect positive- and negative-strand replicon and viral RNAs, are indicated. These probes contain 138 nt of sequence complementary to R2Z and R3Z and 70 nt of sequence complementary to sequences in both replicon and viral RNAs. (B) Scheme for detecting trans encapsidation of replicon RNAs. DNA plasmids that encode either R2Z or R3Z RNAs under the control of a T7 promoter were transfected into KJT7 cells, which constitutively express T7 RNA polymerase. After the R2Z or R3Z RNAs accumulated, cells were superinfected with wild-type or mutant poliovirus. Encapsidation of R2Z or R3Z RNA was detected either by using the cytoplasmic extracts to infect a new monolayer of HeLa cells or by quantifying the amount of replicon RNA in purified virions.
FIG. 2
FIG. 2
Time course of accumulation of R3Z RNA following transfection of KJT7 cells with T7R3Z DNA. RNase protection experiments show the amount of positive-strand R3Z RNA at various times posttransfection (see Materials and Methods). The amount of R3Z RNA in extracts prepared from 4 × 106 KJT7 cells was determined from standard curves of the RNase protection signal from known amounts of R3Z RNA transcripts.
FIG. 3
FIG. 3
trans encapsidation of R2Z and R3Z RNAs. The RNase protection experiment shows the amounts of replicon and viral RNAs produced under various conditions. Total cytoplasmic RNA was extracted from 4 × 106 KJT7 cells that were transfected with plasmids that encode replicon RNAs and superinfected with wild-type poliovirus as indicated. The RNA was then subjected to RNase protection (lanes 6 to 10). The result of using the KJT7 cytoplasmic extracts to infect 4 × 106 HeLa cells for 5 h at 37°C is shown in lanes 11 to 20; only encapsidated RNAs should be capable of initiating infection in the HeLa cells. Lanes that display duplicate experiments are indicated by brackets. RNase protection conditions under which the 76-nt duplex that represents the wild-type RNA was relatively unstable were chosen; thus, the amounts of radioactivity in the “virus” and “replicon” bands cannot be compared directly. However, the use of standard curves of known amounts of replicon and wild-type RNAs (lanes 1 to 5 and data not shown) allowed the determination of the actual amounts of each RNA present in the cytoplasmic extracts.
FIG. 4
FIG. 4
Test for complementation of the guanidine sensitivity of R3Z RNA synthesis by superinfecting 3NC-202guaR virus. KJT7 cells were transfected with R3Z-expressing DNA and incubated for 24 h before being superinfected or mock infected with 3NC-202guaR virus and treated with 0.5 mM guanidine. (A) RNase protection was used to quantify the amount of positive-strand R3Z RNA present in the cytoplasmic extracts. Comparison of the amount of labeled protected RNA to that protected by known amounts of RNA in a standard curve was used to determine the amount of positive-strand R3Z RNA in extracts from 1.6 × 107 cells. Error bars are shown for samples that were tested in duplicate in this particular experiment, one of several independent experiments. (B) The amounts of negative-strand R3Z RNA from 1.6 × 108 cells in the presence and absence of 0.5 mM guanidine and superinfecting 3NC-202guaR virus were analyzed by two-cycle RNase protection (35) and comparison to standard curves.
FIG. 5
FIG. 5
Test of encapsidation of R3Z RNAs in the presence and absence of guanidine treatment and superinfection by 3NC-202guaR virus. (A) Accumulation of R3Z and 3NC-202guaR positive-strand RNAs in the cytoplasm of transfected and superinfected cells in the presence and absence of 0.5 mM guanidine. Lane P contains 0.35 fmol of full-length probe RNA, and lanes 1 to 14 contain labeled probe RNA protected by 5 μg of tRNA (lane 1), known amounts of R3Z RNA to generate a standard curve (lanes 2 to 6), and the cytoplasmic extracts indicated (lanes 7 to 14). Brackets indicate duplicate experiments. (B) Accumulation of R3Z and 3NC-202guaR positive-strand RNAs in HeLa cells infected with extracts from the transfected and superinfected KJT7 cells shown in panel A. HeLa cells were infected with cytoplasmic extracts and incubated for 10 h at 32.5°C, and cytoplasmic extracts were prepared. RNase protection identifies bands protected by R3Z and 3NC-202guaR RNAs. Experiments were performed in duplicate or quadruplicate as indicated by the brackets.
FIG. 6
FIG. 6
Model for coupling between virion assembly and RNA synthesis. Viral RNA strands are hypothesized to be coated with proteins of the viral RNA replication complex on the surface of the double cytoplasmic membranes (52) on which the RNA replication complexes assemble. Interactions between the 14S pentamers, subviral particles that accumulate in poliovirus-infected cells, and the proteins of the replication complex are proposed to position these subviral particles to encapsidate single-stranded RNA as it emerges from the replication complex. The presence of the RNA facilitates the addition of additional pentamers to the growing virion, possibly by increasing the rate of assembly about the threefold symmetry axis (9). Specificity for poliovirus RNA is dictated primarily by the proteins in the RNA replication complex, not by a particular sequence in the viral RNA. This image was also suggested by the electron microscopic images of Pfister et al. (40).
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