Recombinant YFV Nonstructural protein 5 (Fragment) [His]

Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact-dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.

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.