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Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

ABSTRACT: The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsid cleavage site in tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) by the 2A protein of foot-and-mouth disease virus (TBEV-2A and WNV-2A). This obviated the need for NS2B/3 processing at the C terminus of mature protein C while simultaneously producing a 19-amino-acid extension on protein C. Infectious virions were generated with both viruses; the phenotype depended on the host cell. TBEV-2A replicated well in BHK-21 cells but was essentially incapable of replication in tick cells. In contrast, WNV-2A replicated well in mosquito cells but showed a small-plaque phenotype in Vero cells, with frequent production of larger plaques. Sequencing of viral RNA from the larger plaques showed substitutions in the signal sequence for prM, presumably improving coordinated protein processing at the C-prM junction. Furthermore, both TBEV-2A and WNV-2A were also defective in unpackaging and/or early RNA synthesis. Together, these results indicate a role for flavivirus protein C in both viral assembly and RNA replication, possibly by interacting with host cell factors required to set up the cell for RNA replication.

SUBMITTER: Schrauf S

PROVIDER: S-EPMC2772764 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

Schrauf Sabrina S Mandl Christian W CW Bell-Sakyi Lesley L Skern Tim T

Journal of virology 20090819 21

The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsi ...[more]

PMID: 19692461

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Project description:The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. IMPORTANCE:Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs.

Dataset Information

Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

Publications

Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

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