Filoviruses Use the HOPS Complex and UVRAG To Traffic to Niemann-Pick C1 Compartments during Viral Entry.

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.

Transcriptional analysis of viral mRNAs reveals common transcription patterns in cells infected by five different filoviruses.

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses-EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses-in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes.

Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Filoviruses: Interactions with the host cell.

The highly pathogenic filoviruses, Marburg and Ebola virus, are difficult to handle and knowledge of the interactions between filoviruses and their host cells remained enigmatic for many years. Two developments were crucial for the presented advances in our understanding of the cell biology of filoviruses, which is still fragmentary. On the one hand, the number of high containment laboratories increased where handling of the highly pathogenic filoviruses is possible. On the other hand, molecular biological tools have been developed that allow investigation of certain aspects of filoviral replication under normal laboratory conditions which considerably accelerated research on filoviruses. This review describes advances in understanding the interactions between host cells and filoviruses during viral attachment, entry, transcription, assembly and budding.