Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

Rescue and characterization of the first West African Marburg virus 2021 from Guinea.

Marburg virus (MARV) is a causative agent of a severe hemorrhagic fever with high fatality rates endemic in central Africa. Current outbreaks of MARV in Equatorial Guinea and Tanzania underline the relevance of MARV as a public health emergency pathogen. In 2021, the first known human MARV case was confirmed in Guinea, West Africa. Since no infectious virus could be isolated from that fatal case in 2021, we generated recombinant (rec) MARV Guinea by reverse genetics in order to study and characterize this new MARV, which occurred in West Africa for the first time, in terms of its growth properties, detection by antibodies, and therapeutic potential compared to known MARV strains. Our results showed a solid viral replication of recMARV Guinea in human, bat, and monkey cell lines in comparison to other known MARV strains. We further demonstrated that replication of recMARV Guinea in cells can be inhibited by the nucleoside analogue remdesivir. Taken together, we could successfully reconstitute de novo the first West African MARV from Guinea showing similar replication kinetics in cells compared to other central African MARV strains. Our reverse genetics approach has proven successful in characterizing emerging viruses, especially when virus isolates are missing and viral genome sequences are incomplete.

Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase.

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome's very 3'-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60-80. LeaderRNA synthesis requires hexamer phasing in the 3'-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3'-leader promoter. We further found different genomic 3'-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3'-leader promoter, i.e., at the second nucleotide of the genome 3'-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.

Intranasal Administration of a Monoclonal Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection.

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins.

Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.

Analysis of a Putative Late Domain Using an Ebola Virus Transcription and Replication-Competent Virus-Like Particle System.

The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.