The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses.

Poxviruses are unusual DNA viruses that replicate in the cytoplasm. To do so, they encode approximately 100 immunomodulatory proteins that counteract cytosolic nucleic acid sensors such as cGAMP synthase (cGAS) along with several other antiviral response pathways. Yet most of these immunomodulators are expressed very early in infection while many are variable host range determinants, and significant gaps remain in our understanding of poxvirus sensing and evasion strategies. Here, we show that after infection is established, subsequent progression of the viral lifecycle is sensed through specific changes to mitochondria that coordinate distinct aspects of the antiviral response. Unlike other viruses that cause extensive mitochondrial damage, poxviruses sustain key mitochondrial functions including membrane potential and respiration while reducing reactive oxygen species that drive inflammation. However, poxvirus replication induces mitochondrial hyperfusion that independently controls the release of mitochondrial DNA (mtDNA) to prime nucleic acid sensors and enables an increase in glycolysis that is necessary to support interferon stimulated gene (ISG) production. To counter this, the poxvirus F17 protein localizes to mitochondria and dysregulates mTOR to simultaneously destabilize cGAS and block increases in glycolysis. Our findings reveal how the poxvirus F17 protein disarms specific mitochondrially orchestrated responses to later stages of poxvirus replication.

FEZ1 phosphorylation regulates HSPA8 localization and interferon-stimulated gene expression.

Fasciculation and elongation protein zeta-1 (FEZ1) is a multifunctional kinesin adaptor involved in processes ranging from neurodegeneration to retrovirus and polyomavirus infection. Here, we show that, although modulating FEZ1 expression also impacts infection by large DNA viruses in human microglia, macrophages, and fibroblasts, this broad antiviral phenotype is associated with the pre-induction of interferon-stimulated genes (ISGs) in a STING-independent manner. We further reveal that S58, a key phosphorylation site in FEZ1's kinesin regulatory domain, controls both binding to, and the nuclear-cytoplasmic localization of, heat shock protein 8 (HSPA8), as well as ISG expression. FEZ1- and HSPA8-induced changes in ISG expression further involved changes in DNA-dependent protein kinase (DNA-PK) accumulation in the nucleus. Moreover, phosphorylation of endogenous FEZ1 at S58 was reduced and HSPA8 and DNA-PK translocated to the nucleus in cells stimulated with DNA, suggesting that FEZ1 is a regulatory component of the recently identified HSPA8/DNA-PK innate immune pathway.

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome.

Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.

An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses.

An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne's disease-infected animals.

Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage® ) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml-1 ) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low-level bacteraemia is associated with these infections in cattle. In a study of M. bovis-infected cattle (n = 41), the sensitivity of the Actiphage® method was 95 % (95 % CI; 0.84-0.99) and specificity was 100 % (95% CI; 0.92-1). We further used Actiphage® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne's infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection.

Despite producing enormous amounts of cytoplasmic DNA, poxviruses continue to replicate efficiently by deploying an armory of proteins that counter host antiviral responses at multiple levels. Among these, poxvirus protein F17 dysregulates the host kinase mammalian target of rapamycin (mTOR) to prevent the activation of stimulator of interferon genes (STING) expression and impair the production of interferon-stimulated genes (ISGs). However, the host DNA sensor(s) involved and their impact on infection in the absence of F17 remain unknown. Here, we show that cyclic-di-GMP-AMP (cGAMP) synthase (cGAS) is the primary sensor that mediates interferon response factor (IRF) activation and ISG responses to vaccinia virus lacking F17 in both macrophages and lung fibroblasts, although additional sensors also operate in the latter cell type. Despite this, ablation of ISG responses through cGAS or STING knockout did not rescue defects in late-viral-protein production, and the experimental data pointed to other functions of mTOR in this regard. mTOR adjusts both autophagic and protein-synthetic processes to cellular demands. No significant differences in autophagic responses to wild-type or F17 mutant viruses could be detected, with autophagic activity differing across cell types or states and exhibiting no correlations with defects in viral-protein accumulation. In contrast, results using transformed cells or altered growth conditions suggested that late-stage defects in protein accumulation reflect failure of the F17 mutant to deregulate mTOR and stimulate protein production. Finally, rescue approaches suggest that phosphorylation may partition F17's functions as a structural protein and mTOR regulator. Our findings reveal the complex multifunctionality of F17 during infection.IMPORTANCE Poxviruses are large, double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon-stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ∼100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses, we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.

Translational control during poxvirus infection.

Poxviruses are an unusual family of large double-stranded (ds) DNA viruses that exhibit an incredible degree of self-sufficiency and complexity in their replication and immune evasion strategies. Indeed, amongst their approximately 200 open reading frames (ORFs), poxviruses encode approximately 100 immunomodulatory proteins to counter host responses along with complete DNA synthesis, transcription, mRNA processing and cytoplasmic redox systems that enable them to replicate exclusively in the cytoplasm of infected cells. However, like all other viruses poxviruses do not encode ribosomes and therefore remain completely dependent on gaining access to the host translational machinery in order to synthesize viral proteins. Early studies of these intriguing viruses helped discover the mRNA cap and polyadenylated (polyA) tail that we now know to be present on most eukaryotic messages and which play fundamental roles in mRNA translation, while more recent studies have begun to reveal the remarkable lengths poxviruses go to in order to control both host and viral protein synthesis. Here, we discuss some of the central strategies used by poxviruses and the broader battle that ensues with the host cell to control the translation system, the outcome of which ultimately dictates the fate of infection. This article is categorized under: Translation > Translation Regulation.

Poxviruses Evade Cytosolic Sensing through Disruption of an mTORC1-mTORC2 Regulatory Circuit.

Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.