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Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication.

ABSTRACT: The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHHs. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention.

SUBMITTER: Hanke L

PROVIDER: S-EPMC5452021 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication.

Hanke Leo L Schmidt Florian I FI Knockenhauer Kevin E KE Morin Benjamin B Whelan Sean Pj SP Schwartz Thomas U TU Ploegh Hidde L HL

EMBO reports 20170410 6

The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand ...[more]

PMID: 28396572

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Project description:The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an "unstructured" linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function.NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in the pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

Project description:Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

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Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication.

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Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication.

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