Significance: The adenovirus capsid protein is built by three main capsomers: hexon, fiber, and penton base. Entry is mediated by fiber proteins anchoring the virus to host cell receptors and is followed by penton base proteins engaging coreceptors, resulting in entry. Here, we demonstrate that human adenovirus species D types, which constitute two-thirds of all human adenoviruses, enter host cells through a direct interaction between the hexon protein and CD46. This study provides insights into the entry mechanisms used by human adenoviruses. As these viruses are also used as vaccine vectors for prevention of other infectious diseases, the results provided will also be useful for further development of human adenovirus species D types as vaccine vectors.

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an improved haploid screening platform to identify the relevant host factors for BTV infection. Our screening tool identified and validated the host factor Niemann–Pick C1 (NPC1), a lysosomal membrane protein that is involved in lysosomal cholesterol transport, as a critical factor in BTV infection. This finding prompted us to investigate the possibility of testing imipramine, an antidepressant drug known to inhibit NPC1 function by interfering with intracellular cholesterol trafficking. In this study, we evaluated the sensitivity of BTV to imipramine using in vitro assays. Our results demonstrate that imipramine pretreatment inhibited in vitro replication and progeny release of BTV-4, BTV-8, and BTV-16. Collectively, our findings highlight the importance of NPC1 for BTV infection and recommend the reprofiling of imipramine as a potential antiviral drug against BTV.

The virus responsible for COVID-19, SARS-CoV-2, continues to spread through the world. The ongoing emergence of new variants with increased viral transmission and immune evasion continue to pose a challenge. Although large-scale genetic screens have identified numerous host factors required for SARS-CoV-2 infection, however the function of these hits remain incompletely understood. In this study, we performed a haploid forward genetic screen in chemically mutagenized mouse embryonic stem cells overexpressing human ACE2 and identified MED13 and DDX60 as important host factors involved in the modulation of SARS-CoV-2 infection. In this study, we have identified and characterized the function of these key element factors for SARS-CoV2 infection. Knockdown of CKM subunits—with the exception of CDK8—or the helicase DDX60 was sufficient to reduce SARS-CoV-2 infection across Vero E6, A549, and Calu-3 cells. During SARS-CoV-2 infection, MED13 was found to function downstream of the JAK/STAT interferon pathway, but also showed another function independently of the interferon response pathway. Surprisingly, while DDX60 is traditionally involved in the interferon response pathway, its knockdown reduces SARS-CoV-2 infection, suggesting DDX60 can promote SARS-CoV-2 infection Interestingly, while inactivation of MED13 or DDX60 markedly reduced SARS-CoV-2 and SARS-CoV infection, it did not affect MERS-CoV. Collectively, these results identify MED13 and DDX60 as critical host determinants for SARS-CoV-2 related-coronaviruses with pandemic potential. Impact statement: Using an unbiased haploid genetic screening approach, this study identifies for the first time MED13 and DDX60 as host factors that influence coronavirus replication through transcriptional and interferon-associated pathways. While DDX60 has previously been linked to antiviral signaling, our findings suggest it can promote coronavirus infection. Most interestingly, the effects of these factors extend beyond SARS-CoV-2 to other coronaviruses. These findings broaden the current understanding of coronavirus-host interaction by highlighting transcriptional control and interferon-associated pathways as modulators of infection rather than focusing solely on viral entry. The work provides mechanistic insight into how host regulatory networks influence coronavirus replication and suggests potential targets for host-directed therapies with activity against multiple present and future coronavirus threats. Data summary: The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Repositories: The data generated in this study are provided in the Supplementary Information/Source Data file. Sequencing data are available on NCBI Sequence Read Archive under the accession number BioProject PRJNA1271794. The next generation sequencing data generated in this study has been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX29485992, SRX29485993, SRX29485994, SRX29485995, SRX29485996, SRX29485997, SRX29485998, SRX29485999, SRX29486000, SRX29486001, SRX29486002, SRX29486003, SRX29486004, SRX29486005, SRX29486006, SRX29486007, SRX29486010, SRX29486011, SRX29486012, SRX29486013, SRX29486014, SRX29486015, SRX29486016, SRX29486017, SRX29486018, SRX29486019, SRX29486021, SRX29486022, SRX29486023, SRX29486024