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.

YTHDF2 Is Downregulated in Response to Host Shutoff Induced by DNA Virus Infection and Regulates Interferon-Stimulated Gene Expression.

Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

Ribosomes in poxvirus infection.

Poxviruses are large double-stranded DNA viruses that encode their own DNA replication, transcription, and mRNA biogenesis machinery, which underlies their ability to replicate entirely in the cytoplasm. However, like all other viruses, poxviruses remain dependent on host ribosomes to translate their mRNAs into the viral proteins needed to complete their replication cycle. While earlier studies established a fundamental understanding of how poxviruses wrestle with their hosts for control of translation initiation and elongation factors that guide ribosome recruitment and mRNA decoding, recent work has begun to reveal the extent to which poxviruses directly target the ribosome itself. This review summarizes our current understanding of the regulation of ribosomes and translation in poxvirus infection.

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling.

Receptor for activated C kinase 1 (RACK1) is a small ribosomal subunit protein that is phosphorylated by vaccinia virus (VacV) to maximize translation of postreplicative (PR) mRNAs that harbor 5' polyA leaders. However, RACK1 is a multifunctional protein that both controls translation directly and acts as a scaffold for signaling to and from the ribosome. This includes stress signaling that is activated by ribosome-associated quality control (RQC) and ribotoxic stress response (RSR) pathways. As VacV infection activates RQC and stress signaling, whether RACK1 influences viral protein synthesis through its effects on translation, signaling, or both remains unclear. Examining the effects of genetic knockout of RACK1 on the phosphorylation of key mitogenic and stress-related kinases, we reveal that loss of RACK1 specifically blunts the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at late stages of infection. However, RACK1 was not required for JNK recruitment to ribosomes, and unlike RACK1 knockout, JNK inhibitors had no effect on viral protein synthesis. Moreover, reduced JNK activity during infection in RACK1 knockout cells contrasted with the absolute requirement for RACK1 in RSR-induced JNK phosphorylation. Comparing the effects of RACK1 knockout alongside inhibitors of late stage replication, our data suggest that JNK activation is only indirectly affected by the absence of RACK1 due to reduced viral protein accumulation. Cumulatively, our findings in the context of infection add further support for a model whereby RACK1 plays a specific and direct role in controlling translation of PR viral mRNAs that is independent of its role in ribosome-based stress signaling. IMPORTANCE Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal protein that regulates translation directly and mediates signaling to and from the ribosome. While recent work has shown that RACK1 is phosphorylated by vaccinia virus (VacV) to stimulate translation of postreplicative viral mRNAs, whether RACK1 also contributes to VacV replication through its roles in ribosome-based stress signaling remains unclear. Here, we characterize the role of RACK1 in infected cells. In doing so, we find that RACK1 is essential for stress signal activation by ribotoxic stress responses but not by VacV infection. Moreover, although the loss of RACK1 reduces the level of stress-associated JNK activation in infected cells, this is an indirect consequence of RACK1's specific requirement for the synthesis of postreplicative viral proteins, the accumulation of which determines the level of cellular stress. Our findings reveal both the specific role of RACK1 and the complex downstream effects of its control of viral protein synthesis in the context of infection.

Herpesviruses assimilate kinesin to produce motorized viral particles.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.

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.

Human Cytomegalovirus Exploits TACC3 To Control Microtubule Dynamics and Late Stages of Infection.

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. Distinct from other viruses studied to date, at mid- to late stages of its unusually protracted replication cycle, human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure, termed the assembly compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of transforming acidic coiled coil protein 3 (TACC3) to recruit the MT polymerase, chTOG, from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. IMPORTANCE Diverse viruses rely on host cell microtubule networks to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. However, precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the assembly compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid- to late stages of infection, HCMV increases the expression of transforming acidic coiled coil protein 3 (TACC3) to control the localization of the microtubule polymerase, chTOG. This, in turn, enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

Cytoplasmic control of intranuclear polarity by human cytomegalovirus.

Despite its size and rigidity, the cell nucleus can be moved or reorganized by cytoskeletal filaments under various conditions (for example, during viral infection)1-11. Moreover, whereas chromatin organizes into non-random domains12, extensive heterogeneity at the single-cell level13 means that precisely how and why nuclei reorganize remains an area of intense investigation. Here we describe convolutional neural network-based automated cell classification and analysis pipelines, which revealed the extent to which human cytomegalovirus generates nuclear polarity through a virus-assembled microtubule-organizing centre. Acetylation of tubulin enables microtubules emanating from this centre to rotate the nucleus by engaging cytoplasmically exposed dynein-binding domains in the outer nuclear membrane protein nesprin-2G, which polarizes the inner nuclear membrane protein SUN1. This in turn creates intranuclear polarity in emerin, and thereby controls nuclear actin filaments that spatially segregate viral DNA from inactive histones and host DNA, maximizing virus replication. Our findings demonstrate the extent to which viruses can control the nucleus from the cytoplasm.

Proteomic and mechanistic dissection of the poxvirus-customized ribosome.

Ribosomes are often viewed as protein synthesis machines that lack intrinsic regulatory capacity. However, studies have established that ribosomes can functionally diversify through changes in the composition of, or post-translational modifications to ribosomal subunit proteins (RPs). We recently found that poxviruses phosphorylate unique sites in the RP, receptor for activated C kinase 1 (RACK1) to enhance viral protein synthesis. Here, we developed approaches for large-scale proteomic analysis of ribosomes isolated from cells infected with different viruses. Beyond RACK1, we identified additional phosphorylation events within RPS2 and RPS28 that arise during poxvirus infection, but not other viruses tested. The modified sites lie within unstructured loop domains that position around the mRNA entry and exit channel, respectively, and site-substitution mutants revealed that each modified residue contributed differently to poxvirus replication. Our findings reveal the broader extent to which poxviruses customize host ribosomes and provide new insights into how ribosomes can functionally diversify.

TACC3 Regulates Microtubule Plus-End Dynamics and Cargo Transport in Interphase Cells.

End-binding proteins (EBs) are widely viewed as master regulators of microtubule dynamics and function. Here, we show that while EB1 mediates the dynamic microtubule capture of herpes simplex virus type 1 (HSV-1) in fibroblasts, in neuronal cells, infection occurs independently of EBs through stable microtubules. Prompted by this, we find that transforming acid coiled-coil protein 3 (TACC3), widely studied in mitotic spindle formation, regulates the cytoplasmic localization of the microtubule polymerizing factor chTOG and influences microtubule plus-end dynamics during interphase to control infection in distinct cell types. Furthermore, perturbing TACC3 function in neuronal cells resulted in the formation of disorganized stable, detyrosinated microtubule networks and changes in cellular morphology, as well as impaired trafficking of both HSV-1 and transferrin. These trafficking defects in TACC3-depleted cells were reversed by the depletion of kinesin-1 heavy chains. As such, TACC3 is a critical regulator of interphase microtubule dynamics and stability that influences kinesin-1-based cargo trafficking.

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity within a loop domain.

Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved ß-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plant - but not uncharged yeast or human loop - enhances translation of mRNAs with adenosine-rich 5' untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.

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.

Exploitation of Cytoskeletal Networks during Early Viral Infection.

Being dependent upon host transport systems to navigate the cytoplasm, viruses have evolved various strategies to manipulate cytoskeletal functions. Generally, viruses use the actin cytoskeleton to control entry and short-range transport at the cell periphery and exploit microtubules (MTs) for longer-range cytosolic transport, in some cases to reach the nucleus. While earlier studies established the fundamental importance of these networks to successful infection, the mechanistic details and true extent to which viruses usurp highly specialized host cytoskeletal regulators and motor adaptors is only beginning to emerge. This review outlines our current understanding of how cytoskeletal regulation contributes specifically to the early stages of viral infection, with a primary focus on retroviruses and herpesviruses as examples of recent advances in this area.

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.

Author Correction: HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration.

The original version of this Article contained an error in the Methods section 'Viruses and drugs'. The timing for drug treatment of CHME3 4 × 4 or 293T cells with γ-secretase inhibitor or BACE1 inhibitor was incorrectly given as '1 day prior to infection or transfection' and should have stated '4 or 6 h post transfection or infection, respectively'. This error is now corrected in both the PDF and HTML versions of the Article.

ZNF598 Plays Distinct Roles in Interferon-Stimulated Gene Expression and Poxvirus Protein Synthesis.

Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5' poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.

The HCMV Assembly Compartment Is a Dynamic Golgi-Derived MTOC that Controls Nuclear Rotation and Virus Spread.

Human cytomegalovirus (HCMV), a leading cause of congenital birth defects, forms an unusual cytoplasmic virion maturation site termed the "assembly compartment" (AC). Here, we show that the AC also acts as a microtubule-organizing center (MTOC) wherein centrosome activity is suppressed and Golgi-based microtubule (MT) nucleation is enhanced. This involved viral manipulation of discrete functions of MT plus-end-binding (EB) proteins. In particular, EB3, but not EB1 or EB2, was recruited to the AC and was required to nucleate MTs that were rapidly acetylated. EB3-regulated acetylated MTs were necessary for nuclear rotation prior to cell migration, maintenance of AC structure, and optimal virus replication. Independently, a myristoylated peptide that blocked EB3-mediated enrichment of MT regulatory proteins at Golgi regions of the AC also suppressed acetylated MT formation, nuclear rotation, and infection. Thus, HCMV offers new insights into the regulation and functions of Golgi-derived MTs and the therapeutic potential of targeting EB3.

Correction: Poxviruses: Slipping and sliding through transcription and translation.

[This corrects the article DOI: 10.1371/journal.ppat.1006634.].