RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

Abstract

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. (1)(2)(3)(4). Dengue viruses (DENV-1 to -4), the most prevalent of all arthropod-borne viruses, infect approximately 390 million people per year (1); yellow fever virus (YFV), which causes life-threatening disease (5) has reemerged this year in Africa (6); Zika virus (ZIKV) is currently responsible for a pandemic in the Americas that has caused grave concern because of associations with birth defects and Guillain-Barré syndrome (3). KEYWORDS flavivirus, ribosomal proteins, tran...

Biochemistry and Molecular Biology of Flaviviruses

“…236 Indeed, our work has identified a region in the prM-E coding sequences where elongation is exquisitely sensitive to RPLP1 and RPLP2 depletion. 236 As discussed above, flaviviral ORFs also have “slippery sequences” that cause the ribosome to change its reading frame, creating noncanonical proteins. Detailed studies of these noncanonical elongation mechanisms could reveal similar ones operative among specific classes of cellular mRNAs, which may share properties with the flavivirus genome.…”

“…Unfortunately, the flavivirus field has largely ignored the ribosome as a unique host factor. As described above, we identified the ribosomal stalk proteins RPLP1 and RPLP2 as critically important for flavivirus translation, 213,236 and these studies are leading to the identification of cellular mRNAs that share this requirement and eventually to understand the role of these poorly understood proteins. As our view of the ribosome shifts from a monotonic machine to a more modular one, it is likely we will identify new ribosomal components that are exquisitely required by flaviviruses.…”

“…235 Indeed, studies with flaviviruses may shed light into noncanonical modes of translation elongation. 213,236,237 For example, the acidic phosphoproteins RPLP1 and RPLP2 of the ribosomal stalk, which are thought to recruit elongation factors to the ribosome, are exquisitely required for translation of DENV2 RNA but are not required for global cell protein synthesis. 236 Indeed, our work has identified a region in the prM-E coding sequences where elongation is exquisitely sensitive to RPLP1 and RPLP2 depletion.…”

“…213,236,237 For example, the acidic phosphoproteins RPLP1 and RPLP2 of the ribosomal stalk, which are thought to recruit elongation factors to the ribosome, are exquisitely required for translation of DENV2 RNA but are not required for global cell protein synthesis. 236 Indeed, our work has identified a region in the prM-E coding sequences where elongation is exquisitely sensitive to RPLP1 and RPLP2 depletion. 236 As discussed above, flaviviral ORFs also have “slippery sequences” that cause the ribosome to change its reading frame, creating noncanonical proteins.…”

Highlights of the 30th International Conference on Antiviral Research

“…Mariano Garcia-Blanco and colleagues have conducted genome-scale RNAi screens to identify candidate host factors. They identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial host factors required for DENV and YFV infection (Campos et al, 2017). RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk.…”