Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Structure-function analysis of vaccinia virus H7 protein reveals a novel phosphoinositide binding fold essential for poxvirus replication.

UNLABELLED: Phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide binding domains. Recently, a group of poxvirus proteins, termed viral membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven α-helices and a highly curved three-stranded antiparallel ß-sheet. We identified a phosphoinositide binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for viral replication and for binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggest that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis. IMPORTANCE: Poxvirus viral membrane assembly proteins (VMAPs) were recently shown to be essential for poxvirus membrane biogenesis. One of the key components of VMAPs is the H7 protein. However, no known structural motifs could be identified from its sequence, and there are no homologs of H7 outside the poxvirus family to suggest a biochemical function. We have determined the crystal structure of the vaccinia virus (VACV) H7 protein. The structure displays a novel fold with a distinct and positively charged surface. Our data demonstrate that H7 binds phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate and that the basic surface patch is indeed required for phosphoinositide binding. In addition, mutation of positively charged residues required for lipid binding disrupted VACV replication. Phosphoinositides and phosphoinositide binding proteins play critical roles in membrane and protein trafficking in eukaryotes. Our study demonstrates that VACV H7 displays a novel fold for phosphoinositide binding, which is essential for poxvirus replication.

An alphavirus-derived self-amplifying mRNA encoding PpSP15-LmSTI1 fusion protein for the design of a vaccine against leishmaniasis.

The main aims of the present study were to design a fusion protein of Leishmania major stress-inducible protein 1 (LmSTI1) and Phlebotomus papatasi SP15 (PpSP15), and to express it in the form of alphavirus packaged Self-amplifying mRNA (SAM). Two combinations, PpSP15-LmSTI1 and LmSTI1-PpSP15 fusion forms, were analyzed for folding and minimum free energies of the mRNA. Conformational studies on 3D modeled fusion and native forms were performed, and the Root-Mean-Square-distance (RMSD) of the Cα atomic coordinates were calculated. Antigenicity and stability were predicted using bioinformatics tools. The coding sequences of PpSP15-LmSTI1 fusion, PpSP15, and LmSTI1 were cloned into an alphavirus-based vector and used to produce the SAM constructs. All the subcloned constructs were then subjected to packaging in the form of viral replicon particles (VRPs),and were evaluated for their ability to infect BHK-21 cells and express the recombinant fusion proteins. The in-silico analysis indicated that the PpSP15-LmSTI1 combination could be a promising candidate based on lower folding ΔG of mRNA, higher protein antigenicity and lower instability indexes, and less conformational changes compared to the native proteins and the LmSTI1-PpSP15 fusion form. Packaged SAM encoding fusion and native antigens are used for infection of mammalian cells and for recombinant protein expression. This is the first study on in silico designing and successful packaging of an alphavirus-derived SAM in the form of the VRPs to target leishmaniasis.

Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts.

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 ± 0.05 µM, similar to Kd,app values obtained by others. As previously observed, the PLC-δ1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein's capacity to interact with membranes.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.