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The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells
1Department of Cellular, Viral, and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Abstract:
In poliovirus-infected HeLa cells, the mechanism of protein synthesis initiation factor recognition of m7G cap groups on mRNA is impaired. Translation of capped host cell mRNAs is inhibited, whereas translation of uncapped poliovirus mRNA proceeds exclusively. The site of this defect has been localized to the cap-binding protein complex (CBPC). To elucidate the specific structural and functional defects of the CBPC following poliovirus infection, the CBPC and/or its polypeptide components were purified from uninfected and poliovirus-infected HeLa cells. The CBPC from uninfected cells consisted of tightly associated 24- and 220-kDa polypeptides; minor amounts of polypeptides of 40, 44, and 80 kDa also consistently co-purified with the p24/p220 cores. No evidence of a 50-kDa, eIF-4A-related polypeptide subunit of the CBPC was obtained. The CBPC from poliovirus-infected cells had undergone major structural alterations. The 220-kDa component was absent; antigenically related (100-130 kDa) degradation products were present instead. The 24-kDa component co-purified with the p220 degradation products, but other components were missing. The association of the infected cell CBPC components was quite labile compared with that demonstrated by the components of CBPC from uninfected cells. Differential stimulation of capped, but not uncapped mRNAs in a cell-free translation assay was demonstrated by unmodified CBPC. Conversely, modified CBPC from poliovirus-infected cells differentially stimulated in vitro translation of uncapped poliovirus mRNA but not capped mRNAs. The implications of these results for the mechanism of cap-independent translation are briefly discussed.
Insights
Poliovirus infection disrupts the cap-binding protein complex (CBPC) in HeLa cells, inhibiting host mRNA translation while promoting viral mRNA translation. This occurs due to structural alterations in the CBPC, affecting protein synthesis initiation.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Poliovirus infection impairs protein synthesis initiation in host cells.
- This impairment affects the recognition of the m7G cap structure on mRNA.
- Translation of host cell mRNAs is inhibited, while uncapped viral mRNA translation is favored.
Purpose of the Study:
- To investigate the structural and functional defects of the cap-binding protein complex (CBPC) after poliovirus infection.
- To understand the mechanism of cap-independent translation promoted by poliovirus.
Main Methods:
- Purification of CBPC and its polypeptide components from uninfected and poliovirus-infected HeLa cells.
- Analysis of CBPC structural integrity and polypeptide composition.
- Assessment of CBPC function using cell-free translation assays with capped and uncapped mRNAs.
Main Results:
- Uninfected HeLa cell CBPC comprises 24- and 220-kDa polypeptides with stable associations.
- Infected cell CBPC shows major structural alterations: absence of the 220-kDa component, presence of degradation products (100-130 kDa), and labile associations.
- Unmodified CBPC stimulates capped mRNA translation; modified CBPC from infected cells stimulates uncapped viral mRNA translation.
Conclusions:
- Poliovirus infection structurally modifies the CBPC, leading to impaired host mRNA translation.
- The altered CBPC facilitates cap-independent translation of poliovirus mRNA.
- These findings provide insights into the mechanisms of viral translation strategies.