Reprogramming Bacteriophage Host Range through Structure-Guided Design of Chimeric Receptor Binding Proteins.

Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-Å resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility.

Cell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV-induced cell motility, indicating that additional viral factors must be involved. Here, we show that the VACV epidermal growth factor homologue, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10, after which it acts largely in a paracrine manner to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR, MAPK, FAK and ADAM10 inhibitors or with VGF-deleted and F11-deleted viruses revealed defects in radial velocity and directional migration efficiency, leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor-induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

DNA virus uncoating.

Virus genomes are condensed and packaged inside stable proteinaceous capsids that serve to protect them during transit from one cell or host organism, to the next. During virus entry, capsid shells are primed and disassembled in a complex, tightly-regulated, multi-step process termed uncoating. Here we compare the uncoating-programs of DNA viruses of the pox-, herpes-, adeno-, polyoma-, and papillomavirus families. Highlighting the chemical and mechanical cues virus capsids respond to, we review the conformational changes that occur during stepwise disassembly of virus capsids and how these culminate in the release of viral genomes at the right time and cellular location to assure successful replication.

siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor.

Poxvirus genome uncoating is a two-step process. First, cytoplasmic viral cores are activated and early viral genes are expressed. Next, cores are disassembled and the genomes released. This second step depends on an early viral factor(s) that has eluded identification for over 40 years. We used a large-scale, high-throughput RNAi screen directed against vaccinia virus (VACV) to identify the VACV AAA+ ATPase D5 as the poxvirus uncoating factor. We show that the ATPase activity of D5 is required for uncoating. Superresolution microscopy suggests that D5 acts directly at viral cores for genome release. Thus, the putative helicase D5 is a multifunctional protein required for genome uncoating and replication. Additionally, in vivo delivery of anti-D5 siRNAs reduced virus production in a mouse model of VACV infection. These results demonstrate the use of virus-targeting RNAi libraries to investigate viral gene function and suggest therapeutic avenues.

Tracking viral genomes in host cells at single-molecule resolution.

Viral DNA trafficking in cells has large impacts on physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.

Direct identification of ligand-receptor interactions on living cells and tissues.

Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.