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There is increasing recognition of the importance of N 6 -methyladenosine (m 6 A) 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 m 6 A writer, reader, and eraser proteins remains unknown.

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. Herpes simplex virus type I and pseudorabies virus assimilate kinesin from host epithelial cells and repurpose the motor to traffic to the nuclei of neurons in the peripheral nervous system.

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.

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. But 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 in order to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. Yet 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) in order 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.

SUMMARY 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., In Brief While EB proteins are widely studied as master regulators of microtubule plus-end dynamics, Furey et al. report EB-independent regulation of microtubule arrays and cargo trafficking by the transforming acid coiled-coil-containing protein, TACC3. By controlling the formation of detyrosinated stable microtubule networks, TACC3 influences kinesin-1-based sorting of both host and pathogenic cargoes., Graphical Abstract

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.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established

The poxvirus vaccinia virus phosphorylates serine/threonine residues in the human small ribosomal subunit RACK1, converting it to a plant-like state to favour translation of poxvirus mRNAs It has been proposed that ribosomes have the capacity to control mRNA-specific translation by changes in their subunit composition or post-translational modifications. Derek Walsh and colleagues reveal a new way that viruses usurp the cellular machinery to facilitate their replication. They show that poxviruses customize host ribosomes to boost translation of viral mRNAs. The vaccinia virus phosphorylates serine/threonine residues in the ribosomal subunit RACK1, creating a RACK1 loop with a negative charge. This results in the preferential translation of poxvirus mRNAs that carry adenosine repeats in the 5′ UTR, which act as translational enhancers. Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements1. However, beyond differential subunit expression during development2,3, evidence for regulated ribosome specification within individual cells has remained elusive1. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5′ untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase4 confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders4.

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.

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.

SUMMARY 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., Graphical Abstract, In brief How ribosomes functionally diversify to selectively control translation is only beginning to be understood. Rollins et al. show that negative charge in a loop domain of the small subunit ribosomal protein RACK1 increases the swiveling motion of the 40S head and broadens the translational capacity of the human ribosome.

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 β-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.

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.

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.

Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (+TIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.

SummaryStable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs, and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant-negative inhibitor of MT stabilization prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection.

Although viruses require cellular functions to replicate, their absolute dependence upon the host translation machinery to produce polypeptides indispensable for their reproduction is most conspicuous. Despite their incredible diversity, the mRNAs produced by all viruses must engage cellular ribosomes. This has proven to be anything but a passive process and has revealed a remarkable array of tactics for rapidly subverting control over and dominating cellular regulatory pathways that influence translation initiation, elongation, and termination. Besides enforcing viral mRNA translation, these processes profoundly impact host cell-intrinsic immune defenses at the ready to deny foreign mRNA access to ribosomes and block protein synthesis. Finally, genome size constraints have driven the evolution of resourceful strategies for maximizing viral coding capacity. Here, we review the amazing strategies that work to regulate translation in virus-infected cells, highlighting both virus-specific tactics and the tremendous insight they provide into fundamental translational control mechanisms in health and disease.

Poxviruses are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells within discrete compartments termed viral factories. Recent work has shown that the prototypical poxvirus, Vaccinia Virus (VacV) sequesters components of the eukaryotic translation initiation complex eIF4F within viral factories while also stimulating formation of eIF4F complexes. However, the forces that govern these events remain unknown. Here, we show that maximal eIF4F formation requires viral DNA replication and the formation of viral factories, suggesting that sequestration functions to promote eIF4F assembly, and identify the ssDNA-binding protein, I3 as a viral factor that interacts and co-localizes with the eIF4F scaffold protein, eIF4G. Although it did not adversely affect host or viral protein synthesis, I3 specifically mediated the binding of eIF4G to ssDNA. Combined, our findings offer an explanation for the specific pattern and temporal process of eIF4G redistribution and eIF4F complex assembly within VacV-infected cells.

Key Points Although viruses encode many of the functions that are required for viral replication, they are completely reliant on the protein synthesis machinery that is present in their host cells.Recruiting cellular ribosomes to translate viral mRNAs represents a crucial step in the replication of all viruses.To ensure translation of their mRNAs, viruses use a diverse collection of strategies (probably pirated from their cellular hosts) to commandeer key translation factors that are required for the initiation, elongation and termination steps of translation.Viruses also neutralize host defences that seek to incapacitate the translation machinery in infected cells., Viruses rely on the translation machinery of the host cell to produce the proteins that are essential for their replication. Here, Walsh and Mohr discuss the diverse strategies by which viruses subvert the host protein synthesis machinery and regulate the translation of viral mRNAs., Viruses are fully reliant on the translation machinery of their host cells to produce the polypeptides that are essential for viral replication. Consequently, viruses recruit host ribosomes to translate viral mRNAs, typically using virally encoded functions to seize control of cellular translation factors and the host signalling pathways that regulate their activity. This not only ensures that viral proteins will be produced, but also stifles innate host defences that are aimed at inhibiting the capacity of infected cells for protein synthesis. Remarkably, nearly every step of the translation process can be targeted by virally encoded functions. This Review discusses the diverse strategies that viruses use to subvert host protein synthesis functions and regulate mRNA translation in infected cells.

The host cytoskeleton plays a central role in the life cycle of many viruses yet our knowledge of cytoskeletal regulators and their role in viral infection remains limited. Recently, moesin and ezrin, two members of the ERM (Ezrin/Radixin/Moesin) family of proteins that regulate actin and plasma membrane cross-linking and microtubule (MT) stability, have been shown to inhibit retroviral infection. To further understand how ERM proteins function and whether they also influence infection by other viruses, we identified PDZD8 as a novel moesin-interacting protein. PDZD8 is a poorly understood protein whose function is unknown. Exogenous expression of either moesin or PDZD8 reduced the levels of stable MTs, suggesting that these proteins functioned as part of a cytoskeletal regulatory complex. Additionally, exogenous expression or siRNA-mediated knockdown of either factor affected Herpes Simplex Virus type 1 (HSV-1) infection, identifying a cellular function for PDZD8 and novel antiviral properties for these two cytoskeletal regulatory proteins.

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.

Summary 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.

Herpes simplex virus particles that enter the cell do not randomly associate with microtubule filaments, but require plus end–binding proteins EB1, CLIP-170, and dynactin to initiate retrograde transport to the nucleus., Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end–tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170–mediated capture to initiate transport in primary human cells.

Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons.

40S ribosomes are loaded onto capped mRNAs via the multisubunit translation initiation factors eIF3 and eIF4F. While eIF4E is the eIF4F cap recognition component, the eIF4G subunit associates with 40S-bound eIF3. How this intricate process is coordinated remains poorly understood. Here, we identify an eIF3 subunit that regulates eIF4F modification and show that eIF3e is required for inducible eIF4E phosphorylation. Significantly, recruitment of the eIF4E kinase Mnk1 (MAPK signal-integrating kinase 1) to eIF4F depended on eIF3e, and eIF3e was sufficient to promote Mnk1-binding to eIF4G. This establishes a mechanism by which 40S ribosome loading imparts a phosphorylation mark on the cap-binding eIF4F complex that regulates selective mRNA translation and is synchronized by a specific eIF3 subunit.

Epithelial cells are subject to significant mechanical stress and therefore require a structure to protect them from permanent damage. This structure is the cytoskeleton and it is formed by three classes of proteins: microfilaments (principally actin), microtubules (principally tubulin), and intermediate filaments (IFs) (Steinert and Roop, 1988). The IF proteins differ from the actin and tubulin proteins in several important ways. The IFs are a highly diverse family of proteins. In contrast, there are only approximately 6 forms of actin and six forms each of the 2 types of tubulin (α and,β). IF proteins are non-polar, elongated molecules. In contrast, the actin and tubulin proteins are polar and globular molecules. The sizes of the cytoskeletal proteins range from 6 nm (actin) and 10 nm (IFs) to 23 nm (tubulin).

Although microtubules (MTs) frequently form highly dynamic networks, subsets of MTs become stabilized in response to environmental cues and function as specialized tracks for vesicle and macromolecular trafficking. MT stabilization is controlled by specialized plus-end tracking proteins (+TIPs) whose accumulation at the MT ends is facilitated by the end-binding protein, EB1, and regulated by various signaling pathways. As cargoes themselves, viruses are dependent on MTs for their intracellular movement. Although many viruses affect MT organization, the potential contribution of MT stabilization by +TIPs to infection remains unknown. Here we show that early in infection of primary human fibroblasts, herpes simplex virus type 1 (HSV-1) disrupts the centrosome, the primary MT organizing center in many cell types. As infection progresses HSV-1 induces the formation of stable MT subsets through inactivation of glycogen synthase kinase 3beta by the viral Ser/Thr kinase, Us3. Stable MT formation is reduced in cells infected with Us3 mutants and those stable MTs that form cluster around the trans-Golgi network. Downstream of glycogen synthase kinase 3beta, cytoplasmic linker-associated proteins (CLASPs), specialized host +TIPs that control MT formation at the trans-Golgi network and cortical capture, are specifically required for virus-induced MT stabilization and HSV-1 spread. Our findings demonstrate the biological importance of +TIPs to viral infection and suggest that HSV-1 has evolved to exploit the trans-Golgi network as an alternate MT organizing center to facilitate virus spread.

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus.

International audience; Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-β (IFN-β). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Many of the early events in retroviral infection are not well understood, but it is known that the host cytoskeleton and signaling pathways play integral roles in various entry and post-entry processes. Focal adhesion complexes act as sites of integration for both cytoskeletal organization and integrin signaling at the cell surface. Here, we show that talin-1 and vinculin, two interacting proteins that localize in focal adhesions to mediate integrin linkage to the actin cytoskeleton, function during retroviral infection. Transient overexpression of either talin-1 or vinculin reduced the susceptibility of human cells to infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus. In contrast, transient short interfering RNA-mediated knockdown of talin-1 or vinculin increased infection by pseudotyped HIV-1 and simian immunodeficiency virus, demonstrating that the endogenous forms of these proteins also impaired retroviral infection. Talin-1 or vinculin overexpression inhibited infection by retroviruses that entered the cell by either fusion or endocytosis, while analysis of HIV-1 DNA synthesis demonstrated that the block occurred early in infection and prior to the initiation of reverse transcription. Both factors retained antiviral activity in the presence of actin or microtubule depolymerizing agents. Finally, talin-1 and vinculin expression was found to negatively influence tyrosine phosphorylation of paxillin, a major focal adhesion scaffolding protein whose transient knockdown decreased pseudotyped HIV-1 infection. Together, these findings demonstrate that talin-1 and vinculin negatively affect tyrosine phosphorylation of paxillin, a novel positive regulator of HIV-1 infection, and impose an early block to infection by distinct retroviruses.

In the absence of their own translational machinery, all viruses must gain access to host cell ribosomes to synthesize viral proteins and replicate. Ribosome recruitment and scanning of capped host mRNAs is facilitated by the multisubunit eIF (eukaryotic initiation factor) 4F, which consists of a cap-binding protein, eIF4E and an RNA helicase, eIF4A, assembled on a large scaffolding protein, eIF4G. Although inactivated by many viruses to inhibit host translation, a growing number of DNA viruses are being found to employ diverse strategies to stimulate eIF4F activity in infected cells and maximize viral protein synthesis. These strategies include stimulation of cellular mTOR (mammalian target of rapamycin) signalling to inactivate 4E-BPs (eIF4E-binding proteins), a family of translational repressors that limit eIF4E availability and eIF4F complex formation, together with modulating the activity of the eIF4E kinase Mnk (mitogen-activated protein kinase signal-integrating kinase) in a variety of manners to regulate both host and viral mRNA translation. In some cases, specific viral proteins that mediate these signalling events have been identified, whereas others have been shown to interact with host translation initiation factors or complexes and modify their activity and/or subcellular localization. The present review outlines current understanding of the role of eIF4F in the life cycle of various DNA viruses and discusses its potential as a therapeutic target to suppress viral infection.

The eukaryotic initiation factor eIF4F recruits ribosomes to capped mRNAs while eIF2 mediates start codon recognition to initiate protein synthesis. Increasing interest in targeting translation to suppress tumor growth has led to the development of new classes of inhibitors, including 4EGi-1, which disrupts eIF4F complexes. However, the full effects of this inhibitor and its potential uses in the treatment of other disease states remain unclear. Here, we show that overall rates of protein synthesis in primary human cells were affected only modestly by eIF4F disruption using the mTOR inhibitor Torin1, yet were highly sensitive to 4EGi-1. Translational suppression occurred even at concentrations of 4EGi-1 that were below those required to significantly alter eIF4F levels but were instead found to increase the association of ribosomal complexes containing inactive eIF2α. Although highly stable in culture, the effects of 4EGi-1 on both cellular protein synthesis and ribosome association were readily reversible upon inhibitor removal. In addition, despite potently inhibiting translation, prolonged exposure to 4EGi-1 had only modest effects on cell morphology and protein abundance without affecting viability or stress tolerance to any significant degree, although differential effects on heat shock protein (hsp) expression highlighted distinct 4EGi-1-sensitive modes of hsp induction. In contrast, 4EGi-1 potently suppressed poxvirus replication as well as both reactivation and lytic phases of herpesvirus infection. These findings identify a novel way in which 4EGi-1 affects the host cell's protein synthesis machinery and demonstrate its potential as a noncytotoxic inhibitor of diverse forms of viral infection.

In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host–cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate., Author Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is an important human pathogen and, like all herpesviruses, establishes a state of permanent residency in the infected host called latency. Major sites of KSHV latency are cells of the immune system and cells lining blood vessels. In individuals with weakened immunity, inappropriate growth of these cells driven by the resident virus can give rise to primary effusion lymphoma and Kaposi's sarcoma, respectively. These life-threatening cancers are most common in patients with HIV/AIDS and have become a major source of mortality in parts of sub-Saharan Africa. Under appropriate stimuli, herpesviruses change their relationship with the host cell and begin to manufacture proteins required to assemble new infectious virus particles that can be released and spread. To achieve this, the virus hijacks key processes within the cell and conscripts them into producing viral proteins. In this study, we describe for the first time how KSHV carefully manipulates the host protein synthesis machinery during the switch from latency to this specialized infectious virus production mode. Our results show that although overall protein synthesis is diminished, key components of the host's protein manufacturing machinery are actually stimulated, presumably to accelerate viral protein production.

How vaccinia virus (VV) regulates assembly of the host translation initiation complex eIF4F remains unclear. Here, we show that VV activated host PI3K to stimulate downstream mammalian target of rapamycin (mTOR), a kinase that inactivates the translational repressor 4E-BP1. However, although the mTOR inhibitor rapamycin suppressed VV-induced inactivation of 4E-BP1, it failed to inhibit eIF4F assembly. In contrast, PI3K inhibition in VV-infected cells increased the abundance of hypophosphorylated 4E-BP1 and disrupted eIF4F complex formation. PI3K signaling, therefore, plays a critical role in regulating protein production during VV infection, at least in part by controlling the abundance and activity of 4E-BP1.