Flavivirus internalization is regulated by a size-dependent endocytic pathway.

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.

RNASEK is required for internalization of diverse acid-dependent viruses.

Viruses must gain entry into cells to establish infection. In general, viruses enter either at the plasma membrane or from intracellular endosomal compartments. Viruses that use endosomal pathways are dependent on the cellular factors that control this process; however, these genes have proven to be essential for endogenous cargo uptake, and thus are of limited value for therapeutic intervention. The identification of genes that are selectively required for viral uptake would make appealing drug targets, as their inhibition would block an early step in the life cycle of diverse viruses. At this time, we lack pan-antiviral therapeutics, in part because of our lack of knowledge of such cellular factors. RNAi screening has begun to reveal previously unknown genes that play roles in viral infection. We identified dRNASEK in two genome-wide RNAi screens performed in Drosophila cells against West Nile and Rift Valley Fever viruses. Here we found that ribonuclease kappa (RNASEK) is essential for the infection of human cells by divergent and unrelated positive- and negative-strand-enveloped viruses from the Flaviviridae, Togaviridae, Bunyaviridae, and Orthomyxoviridae families that all enter cells from endosomal compartments. In contrast, RNASEK was dispensable for viruses, including parainfluenza virus 5 and Coxsackie B virus, that enter at the plasma membrane. RNASEK is dispensable for attachment but is required for uptake of these acid-dependent viruses. Furthermore, this requirement appears specific, as general endocytic uptake of transferrin is unaffected in RNASEK-depleted cells. Therefore, RNASEK is a potential host cell Achilles' heel for viral infection.

Virus-induced translational arrest through 4EBP1/2-dependent decay of 5'-TOP mRNAs restricts viral infection.

The mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV), is a highly successful pathogen for which there are no vaccines or therapeutics. Translational arrest is a common antiviral strategy used by hosts. In response, RVFV inhibits two well-known antiviral pathways that attenuate translation during infection, PKR and type I IFN signaling. Despite this, translational arrest occurs during RVFV infection by unknown mechanisms. Here, we find that RVFV infection triggers the decay of core translation machinery mRNAs that possess a 5'-terminal oligopyrimidine (5'-TOP) motif in their 5'-UTR, including mRNAs encoding ribosomal proteins, which leads to a decrease in overall ribosomal protein levels. We find that the RNA decapping enzyme NUDT16 selectively degrades 5'-TOP mRNAs during RVFV infection and this decay is triggered in response to mTOR attenuation via the translational repressor 4EBP1/2 axis. Translational arrest of 5'-TOPs via 4EBP1/2 restricts RVFV replication, and this increased RNA decay results in the loss of visible RNA granules, including P bodies and stress granules. Because RVFV cap-snatches in RNA granules, the increased level of 5'-TOP mRNAs in this compartment leads to snatching of these targets, which are translationally suppressed during infection. Therefore, translation of RVFV mRNAs is compromised by multiple mechanisms during infection. Together, these data present a previously unknown mechanism for translational shutdown in response to viral infection and identify mTOR attenuation as a potential therapeutic avenue against bunyaviral infection.

Instrument-Free Point-of-Care Molecular Detection of Zika Virus.

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.

Potential electrostatic interactions in multiple regions affect human metapneumovirus F-mediated membrane fusion.

The recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F(2) domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.

Sirtuin Inhibitors Are Broadly Antiviral against Arboviruses.

Arthropod-borne viruses are diverse pathogens and are often associated with human disease. These viruses span multiple genera, including flaviviruses, alphaviruses, and bunyaviruses. In a high-throughput drug screen, we found that tenovin-1 was antiviral against the flaviviruses Zika virus and dengue virus. Tenovin-1 is a sirtuin inhibitor, and here we found that inhibition of sirtuins, but not inhibition of the related histone deacetylases, is potently antiviral against diverse arboviruses. Sirtuin inhibitors block infection of arboviruses in multiple human cell types. We found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. However, sirtuin inhibitors had no impact on the replication of flaviviral replicons, suggesting a defect in the establishment of replication. Consistent with this, we found that sirtuin inhibitors impacted double-stranded RNA (dsRNA) accumulation during flaviviral infection. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.IMPORTANCE Arthropod-borne viruses are diverse pathogens and are associated with human disease. Through high-throughput drug screening, we found that sirtuin inhibitors are potently antiviral against diverse arboviruses, including flaviviruses such as West Nile virus, bunyaviruses such as Rift Valley fever virus, and alphaviruses such as chikungunya virus. Sirtuin inhibitors block infection of these viruses in multiple human cell types. Moreover, we found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.

Screening Bioactives Reveals Nanchangmycin as a Broad Spectrum Antiviral Active against Zika Virus.

Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2,000 bioactive compounds for their ability to block Zika virus infection in three distinct cell types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA-approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from infection with receptor tyrosine kinase inhibitors, while placental-derived cells that lack AXL expression are insensitive to this inhibition. Importantly, we identified nanchangmycin as a potent inhibitor of Zika virus entry across all cell types tested, including physiologically relevant primary cells. Nanchangmycin also was active against other medically relevant viruses, including West Nile, dengue, and chikungunya viruses that use a similar route of entry. This study provides a resource of small molecules to study Zika virus pathogenesis.