Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells. Dengue is the most common arthropod-borne viral infection and is spreading to new areas every year. Its causative agent, dengue virus (DENV), has immunologically distinct serotypes that increase the risk of life-threatening diseases such as dengue hemorrhagic fever. However, an effective medication for dengue has not yet been established. There is, therefore, an urgent need to develop new antivirals and vaccines against DENV. Here, we have characterized C19orf66, named Repressor of yield of DENV (RyDEN), as a cellular gene that inhibits the replication of all DENV serotypes. The expression of RyDEN was found to be upregulated by interferon (IFN) treatment and played a critical role in the IFN-mediated anti-DENV response. We also found that RyDEN was likely to block the protein translation of DENV RNA through its association with cellular mRNA-binding proteins and viral RNA. Intriguingly, replication of several other viruses, such as hepatitis C virus, Kunjin virus, and Chikungunya virus, was also limited by RyDEN expression. Thus, this study describes a novel mechanism of an IFN-inducible inhibitory factor for DENV and provides the basis for future development of broad-spectrum antivirals against infectious viral diseases, including dengue.

Flavivirus consists of a large number of arthropod-borne viruses, many of which cause life-threatening diseases in humans. A characteristic feature of flavivirus infection is to induce the rearrangement of intracellular membrane structure in the cytoplasm. This unique membranous structure called replication organelle is considered as a microenvironment that provides factors required for the activity of the flaviviral replication complex. The replication organelle serves as a place to coordinate viral RNA amplification, protein translation, and virion assembly and also to protect the viral replication complex from the cellular immune defense system. In this review, we summarize the current understanding of how the formation and function of membrane-associated flaviviral replication organelle are regulated by cellular factors.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is a major public health concern, but the mechanisms underlying its viral particle formation are not well understood. In this study, we established a system for producing virus-like particles (VLPs) by expressing four structural proteins that make up SARS-CoV-2 virus particles in cells and used a spike (S) protein fused with the HiBiT peptide as a marker for evaluating VLP production. Using this system, we confirmed that the E protein plays an important role in VLP release. Both the co-expression of VPS4A K173Q and ORF3A and treatment with bafilomycin A1 enhanced VLP release. These results suggest that VLPs are released in an endosomal sorting complex required for transport-independent manner and that lysosomal dysfunction is required for the efficient release of VLPs. Screening various E protein mutants revealed that the F56/Y57/Y59 amyloidization motif and the D72/L73/L74/V75 PDZ-binding motif (PBM) are critical for E protein function in VLP release. We also found that E protein expression led to an increase in the pH of lysosomes and that the N15 residue required for viroporin activity, the C40/C43 consensus sequence, or the K63 dibasic motif are required for its function. However, amyloidization or PBM mutations did not affect lysosomal deacidification, suggesting that the mechanisms of E protein activity during VLP formation and lysosomal deacidification are distinct. Overall, this study highlights the importance of the E protein in SARS-CoV-2 viral particle formation, and the results may be useful in the development of drugs that inhibit this process. IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.