Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection

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...

mentioning

confidence: 99%

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

Campos

Wong

Xie

et al. 2017

“…This mode of action is similar to what was previously observed for E1A and CtBP (30), but with repression being the outcome rather than activation, as was the case with CtBP. Interestingly, the insect homologue of RuvBL1 was also shown to be a restriction factor for replication in diverse vector-borne viruses (including West Nile virus, Sindbis virus, dengue virus, Rift Valley fever virus, and vesicular stomatitis virus), suggesting that modulation of RuvBL1 activities may be a common strategy of viral pathogens in order to promote their replication (36). RuvBL1 has a well-established role in cancer and cellular transformation (reviewed in reference 37).…”

Section: + Ifnα2amentioning

confidence: 99%

Suppression of Type I Interferon Signaling by E1A via RuvBL1/Pontin

Olanubi

Frost

Radko

et al. 2017

Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling significantly reduces or halts the ability of a virus to replicate; therefore, viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting the cellular response to interferon. Here, we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon-stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, its depletion had no effect on the ability of the virus to replicate its genome or to drive cells into S phase. E1A was found to bind to RuvBL1 via the C terminus of E1A, and this interaction was important for suppression of interferon-stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here, we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress the interferon response.IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate-early gene E1A was previously shown to affect interferon signaling in several different ways. Here, we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon-stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon and identifies a novel target used by adenovirus for modulation of the cellular interferon pathway. KEYWORDS E1A, Pontin, RuvBL1, adenovirus, interferon H uman adenovirus (HAdV) infects and replicates in terminally differentiated cells, usually of the epithelium (1). In order to replicate within the infected cell, the virus needs to reprogram the intracellular environment to be more permissive to replication of the viral genome. This is mainly due to the type of cell that the virus infects, which is a terminally differentiated epithelial cell lacking in proteins and cofactors required for large-scale DNA replication (2). In addition to cellular reprogramming, the virus needs to be able to hide within the infected cell long enough to accomplish its replicative cycle and spread to neighboring cells. Adenoviruses have evolved several different strategies to suppress the innate and acquired immune systems and prevent the detection and killing of an infected cell. One of the major co...

“…However, some cellular factors could act as CIRFs restricting virus replication (22,83,84). Among the several CIRFs against tombusviruses, Rsp5p and the WW-domain proteins are strong inhibitors, which are present in both yeast and plant cells (64,65).…”

Section: Rsp5p and Ww-domain Proteins Act As Cirfs Against Tombusvirumentioning

confidence: 99%

Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

Barajas

Kovalev

Qin

et al. 2015

Replication of (؉)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WWdomain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WWdomain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCEReplication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus, and it possibly has a regulatory function during viral replication in infected cells. P lus-stranded (ϩ)RNA viruses, which are widespread and emerging pathogens, replicate in the cytosol of infected cells by assembling membrane-bound viral replicase complexes (VRCs). The VRCs consist of the viral RNA and viral proteins, as well as co-opted host-coded proteins (1-8). Rapid progress has recently been made in understanding the functions of the viral replication proteins, including the v...