SMAD proteins: Mediators of diverse outcomes during infection.

Understanding the relationship between host and pathogen is key to combatting disease. SMAD transcription factors, which transmit TGF-ß superfamily signalling, mediate an array of outcomes during embryogenesis, inflammation, cancer, and immunity. Surprisingly, these activities can sometimes be directly opposed; for example, SMAD3 has been reported as tumour suppressor by arresting cell cycle progression but conversely promotes cancer metastasis. A growing body of literature has identified SMADs as prominent targets during viral and bacterial infection for modulating host signalling. During infection, the activity of SMAD-containing transcriptional complexes can be finely tuned by pathogens to enhance infectivity and spread. SMAD signalling can be modulated at many levels, such as upstream at the ligand and receptor, or by direct interactions with SMADs. These alterations can increase pathogen dissemination, induce fibrosis, over-activate, or attenuate the host immune response. Here, we summarise the diverse mechanisms by which pathogens have evolved to sway SMAD signalling in their favour. Understanding the intricacies of host-pathogen interactions through this lens may elucidate aspects of SMAD function in cancer development, homoeostasis, and immune signalling previously overlooked. These insights are an opportunity to identify novel TGF-ß or BMP-targeted therapeutics for applications to infectious disease contexts.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells. We demonstrate that viperin upregulation by HSV-1 infection is cell-type-specific, with mouse cells typically showing greater increases compared with those of human origin. Further, overexpression and knockout of mouse, but not human viperin significantly impedes and increases HSV-1 replication, respectively. In primary mouse fibroblasts, viperin upregulation by infection requires viral gene transcription and occurs in a predominantly IFN-independent manner. Further we identify the N-terminal domain of viperin as being required for the anti-HSV-1 activity. Interestingly, this is the region of viperin that differs most between mouse and human, which may explain the apparent species-specific activity against HSV-1. Finally, we show that HSV-1 virion host shutoff (vhs) protein is a key viral factor that antagonises viperin in mouse cells. We conclude that viperin can be upregulated by HSV-1 in mouse and human cells, and that mouse viperin has anti-HSV-1 activity.

Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9.

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses. Graphic abstract: Pipeline for Cas9 selection of recombinant poxviruses.

Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool.

In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks.

Cell-to-cell spread of vaccinia virus is promoted by TGF-ß-independent Smad4 signalling.

The induction of Smad signalling by the extracellular ligand TGF-ß promotes tissue plasticity and cell migration in developmental and pathological contexts. Here, we show that vaccinia virus (VACV) stimulates the activity of Smad transcription factors and expression of TGF-ß/Smad-responsive genes at the transcript and protein levels. Accordingly, infected cells share characteristics to those undergoing TGF-ß/Smad-mediated epithelial-to-mesenchymal transition (EMT). Depletion of the Smad4 protein, a common mediator of TGF-ß signalling, results in an attenuation of viral cell-to-cell spread and reduced motility of infected cells. VACV induction of TGF-ß/Smad-responsive gene expression does not require the TGF-ß ligand or type I and type II TGF-ß receptors, suggesting a novel, non-canonical Smad signalling pathway. Additionally, the spread of ectromelia virus, a related orthopoxvirus that does not activate a TGF-ß/Smad response, is enhanced by the addition of exogenous TGF-ß. Together, our results indicate that VACV orchestrates a TGF-ß-like response via a unique activation mechanism to enhance cell migration and promote virus spread.

CRISPR/Cas9-Based Genome Editing of HSV.

The CRISPR/Cas9 gene editing system is a robust and versatile technology that has revolutionized our capacity for genome engineering and is applicable in a wide range of organisms, including large dsDNA viruses. Here we provide an efficient methodology that can be used both for marker-based and for marker-free CRISPR/Cas9-mediated editing of the HSV-1 genome. In our method, Cas9, guide RNAs and a homology-directed repair template are provided to cells by cotransection of plasmids, followed by introduction of the HSV genome by infection. This method offers a great deal of flexibility, facilitating editing of the HSV genome that spans the range from individual nucleotide changes to large deletions and insertions.