The artificial intelligence-based structure prediction program AlphaFold-Multimer enabled structural modelling of protein complexes with unprecedented accuracy. Increasingly, AlphaFold-Multimer is also used to discover new protein–protein interactions (PPIs). Here, we present AlphaPulldown, a Python package that streamlines PPI screens and high-throughput modelling of higher-order oligomers using AlphaFold-Multimer. It provides a convenient command-line interface, a variety of confidence scores and a graphical analysis tool. AlphaPulldown is freely available at https://www.embl-hamburg.de/AlphaPulldown. Supplementary note is available at Bioinformatics online.

In common with all segmented negative-sense RNA viruses, bunyavirus transcripts contain heterologous sequences at their 5' termini originating from capped host cell RNAs. These heterologous sequences are acquired by a so-called cap-snatching mechanism. Whereas for nuclear replicating influenza virus the source of capped primers as well as the cap-binding and endonuclease activities of the viral polymerase needed for cap snatching have been functionally and structurally well characterized, our knowledge on the expected counterparts of cytoplasmic replicating bunyaviruses is still limited and controversial. This review focuses on the cap-snatching mechanism of bunyaviruses in the light of recent structural and functional data.

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase. Segmented negative strand viruses (sNSV) such as Influenza, Lassa or Hantaan viruses are responsible for a large number of severe human infectious diseases. Currently, there are vaccines and antiviral treatments available for influenza but none for the infections caused by other sNSV. All carry out transcription by the cap-snatching mechanism, which requires the action of a metal ion dependent endonuclease (EN), a domain within their large viral polymerases. Here we provide the crystal structure of the Hantaan virus (family Bunyaviridae) and Lassa virus (family Arenaviridae) cap-snatching ENs in complex with manganese and a comparative functional study of their catalytic activity. Despite the high structural homology between the two ENs a few changes in the active site, involving a catalytic histidine, cause a different binding of the metal ions with dramatic consequences for their in vitro activity. Hantaan EN binds the metal ions as Influenza A (family Orthomyxoviridae) and LACV (family Bunyaviridae) ENs and all three are active in vitro. In contrast Lassa virus EN is inactive in the same experimental conditions. We can now classify sNSV into two functionally distinct groups (His+ and His- ENs), providing a broad view of the sNSV cap-snatching ENs properties that will be determinant for the comprehensive development of broad-spectrum antiviral drugs. These results also have implications for the viral transcription regulation in the light of recent studies on full-length sNSV polymerases.

Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions.

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein–RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

Hantaviruses infect a wide range of hosts including insectivores and rodents and can also cause zoonotic infections in humans, which can lead to severe disease with possible fatal outcomes. Hantavirus outbreaks are usually linked to the population dynamics of the host animals and their habitats being in close proximity to humans, which is becoming increasingly important in a globalized world. Currently there is neither an approved vaccine nor a specific and effective antiviral treatment available for use in humans. Hantaviruses belong to the order Bunyavirales with a tri-segmented negative-sense RNA genome. They encode only five viral proteins and replicate and transcribe their genome in the cytoplasm of infected cells. However, many details of the viral amplification cycle are still unknown. In recent years, structural biology methods such as cryo-electron tomography, cryo-electron microscopy, and crystallography have contributed essentially to our understanding of virus entry by membrane fusion as well as genome encapsidation by the nucleoprotein. In this review, we provide an update on the hantavirus replication cycle with a special focus on structural virology aspects.