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Analysis of RNA synthesis of type 1 poliovirus by using an in vitro molecular genetic approach
Abstract:
Membranous crude replication complexes (CRC) were isolated from poliovirus-infected HeLa cells as recently described (N. Takeda, R.J. Kuhn, C.-F. Yang, T. Takegami, and E. Wimmer, J. Virol. 60:43-53, 1986). Viruses used to produce the CRC were poliovirus type 1 (Mahoney), [PV-1(M)], poliovirus type 1 (Sabin) [PV-1(S)], and four in vitro recombinants that were constructed from infectious cDNA clones. RNA synthesis in CRC was studied. No end-linked, full-length double-stranded poliovirus RNA was detected in CRC regardless of whether nonionic detergent (Nonidet P-40) was added prior to incubation. Synthesis of VPg-pU and VPg-pUpU, two nucleotidyl proteins presumed to be involved in the initiation of RNA synthesis, was slower at 30 degrees C in CRC induced by PV-1(S) than by PV-1(M). This observation was used to design a pulse-chase experiment whose result suggested that synthesis of VPg-pUpU occurred by uridylylation of VPg-pU. Synthesis of VPg-pU(pU) was thermosensitive in CRC induced by PV-1(S). With CRC of recombinant viruses, the thermosensitive block covaried to nucleotide substitutions in PV-1(S) that mapped to the virus-induced RNA polymerase 3Dpol. We conclude that plus-stranded RNA synthesis in CRC does not proceed via hairpin structures. The results of VPg-pU----VPg-pUpU synthesis are consistent with a model in which VPg-pU is the primer of RNA synthesis mediated by 3Dpol. The data suggest that uridylylation of VPg or a precursor thereof may be catalyzed by 3Dpol itself, a mechanism resembling events occurring in adenovirus DNA replication.
Insights
Poliovirus RNA synthesis initiation involves specific protein-nucleotide intermediates. The study reveals that the viral RNA polymerase (3Dpol) likely catalyzes VPg uridylylation, a key step in initiating viral RNA replication.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Membranous crude replication complexes (CRC) are essential for poliovirus RNA synthesis.
- Understanding the initiation of viral RNA synthesis is crucial for comprehending viral replication.
- Previous studies have described the isolation of CRC from poliovirus-infected cells.
Purpose of the Study:
- To investigate the mechanism of poliovirus RNA synthesis within CRC.
- To identify the roles of specific protein-nucleotide intermediates in RNA synthesis initiation.
- To elucidate the function of the viral RNA polymerase (3Dpol) in this process.
Main Methods:
- Isolation of membranous crude replication complexes (CRC) from poliovirus-infected HeLa cells.
- Study of RNA synthesis in CRC using poliovirus type 1 strains (Mahoney and Sabin) and recombinant viruses.
- Pulse-chase experiments to analyze the synthesis and modification of VPg-linked uridylylated nucleotides.
- Thermosensitivity analysis of replication complexes to map functional domains.
Main Results:
- No end-linked, full-length double-stranded poliovirus RNA was detected in CRC.
- Synthesis of VPg-pU and VPg-pUpU was slower in PV-1(S) CRC compared to PV-1(M) CRC at 30°C.
- Pulse-chase experiments indicated that VPg-pUpU is synthesized by uridylylation of VPg-pU.
- A thermosensitive block in VPg-pU(pU) synthesis in PV-1(S) CRC covaried with mutations in 3Dpol.
- Poliovirus RNA synthesis does not proceed via hairpin structures.
Conclusions:
- VPg-pU serves as the primer for RNA synthesis mediated by the viral RNA polymerase 3Dpol.
- The viral RNA polymerase 3Dpol likely catalyzes the uridylylation of VPg or its precursor.
- This mechanism of initiation resembles events observed in adenovirus DNA replication.