The ubiquitin system has emerged as a master regulator of many, if not all, cellular functions. With its large repertoire of conjugating and ligating enzymes, the ubiquitin system holds a unique mechanism to provide selectivity and specificity in manipulating protein function. As intracellular parasites viruses have evolved to modulate the cellular environment to facilitate replication and subvert antiviral responses. Poxviruses are a large family of dsDNA viruses with large coding capacity that is used to synthetise proteins and enzymes needed for replication and morphogenesis as well as suppression of host responses. This review summarises our current knowledge on how poxvirus functions rely on the cellular ubiquitin system, and how poxviruses exploit this system to their own advantage, either facilitating uncoating and genome release and replication or rewiring ubiquitin ligases to downregulate critical antiviral factors. Whilst much remains to be known about the intricate interactions established between poxviruses and the host ubiquitin system, our knowledge has revealed crucial viral processes and important restriction factors that open novel avenues for antiviral treatment and provide fundamental insights on the biology of poxviruses and other virus families.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MV are further wrapped and secreted to spread infection within the host. However, late during infection MV accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titres. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate-limiting for EV production. The A26 gene is conserved within vertebrate poxviruses, but strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfil different functions represents a novel level of complexity in mammalian viruses with major impact on evolution, adaptation and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intra-host dissemination and inter-host transmission, so virus maturation pathways must be tightly controlled. Here we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation towards the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses but those transmitted exclusively via biting arthropods reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Cells are equipped to defend themselves from invading pathogens through sensors such as cGAS, which upon binding DNA induces type I interferon (IFN) expression. Whilst IFNs are crucial for limiting viral infection and activating adaptive immunity, uncontrolled production causes excessive inflammation and autoimmunity. cGAS binds DNA of both pathogenic and cellular origin and its activity is therefore tightly regulated. This is particularly apparent during mitosis, where cGAS association with chromatin following nuclear membrane dissolution and phosphorylation by mitotic kinases negatively regulate enzymatic activity. Here we describe a novel mechanism by which DNA sensing and other innate immune pathways are regulated during cell division, dependent on cyclin dependent kinases (CDK) 4 and 6. Inhibition of CDK4/6 using chemical inhibitors, shRNA-mediated depletion, or overexpression of cellular CDK4/6 inhibitor p16INK4a, greatly enhanced DNA- or cGAMP-induced expression of cytokines and IFN-stimulated genes (ISG). Mechanistically, CDK4/6-dependent inhibition mapped downstream of cytoplasmic signalling events including STING and IRF3 phosphorylation, limiting innate immune induction at the level of IFNβ mRNA expression. This regulation was universal, occurring in primary and transformed cells of human and murine origin, and broad, as IFNβ expression was inhibited in a CDK4/6-dependent manner downstream of multiple pattern recognition receptors. Together these findings demonstrate that host innate responses are limited by multiple mechanisms during cell division, thus defining cellular replication as an innate immune privileged process that may be necessary to avoid aberrant self-recognition and autoimmunity.

Type 1 interferons (IFN-1) are pleiotropic cytokines with well-established anticancer and antiviral properties, particularly in mucosal tissues. Hence, natural IFN-1-inducing treatments are highly sought after in the clinic. Here, we report for the first time that cryptolepine, a pharmacoactive alkaloid in the medicinal plant Cryptolepis sanguinolenta, is a potent IFN-1 pathway inducer. Cryptolepine increased the transcript levels of JAK1, TYK2, STAT1, STAT2, IRF9, and OAS3, as well as increased the accumulation of STAT1 and OAS3 proteins, similar to recombinant human IFN-α. Cryptolepine effects were observed in multiple cell types including a model of human macrophages. This response was maintained in MAVS and STING-deficient cell lines, suggesting that cryptolepine effects are not mediated by nucleic acids released upon nuclear or organelle damage. In agreement, cryptolepine did not affect cell viability in concentrations that triggered potent IFN-1 activation. In addition, we observed no differences in the presence of a pharmacological inhibitor of TBK1, a pleiotropic kinase that is a converging point for Toll-like receptors (TLRs) and nucleic acid sensors. Together, our results demonstrate that cryptolepine is a strong inducer of IFN-1 response and suggest that cryptolepine-based medications such as C. sanguinolenta extract could be potentially tested in resource-limited regions of the world for the management of chronic viral infections as well as cancers.