PARP14 and PARP9/DTX3L regulate interferon-induced ADP-ribosylation.

PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities.

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection.

Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.

Crossroads in virology: current challenges and future perspectives in the age of emerging viruses.

Ongoing global health challenges posed by emerging and re-emerging viruses have highlighted the critical importance of understanding virus-host interactions in countering these threats. Environmental changes, urbanisation and ecological disruption, coupled with the adaptable nature of viruses, facilitates the emergence and spread of new viruses. This Editorial emphasises the urgency of a concerted effort in understanding virus-host interactions to inform the development of therapeutics and vaccines, and help predict disease outcomes. Furthermore, efforts to monitor viral evolution, identify mutations of concern, and develop 'universal' vaccines and broad-spectrum antiviral drugs are needed to counter viral evolution and potentially prevent future viral emergences. Widespread public mistrust surrounding viruses and vaccines also calls for improvement in science communication. A 'One Health' approach that advocates the development of robust global health systems, interdisciplinary collaborations and equity in health access is therefore imperative for transforming the virology landscape.

Updated protein domain annotation of the PARP protein family sheds new light on biological function.

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles.

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.

Mutation nsp6 L232F associated with MERS-CoV zoonotic transmission confers higher viral replication in human respiratory tract cultures ex-vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes zoonotic disease. Dromedary camels are the source of zoonotic infection. We identified a mutation of amino acid leucine to phenylalanine in the codon 232 position of the non-structural protein 6 (nsp6) (nsp6 L232F) that is repeatedly associated with zoonotic transmission. We generated a pair of isogenic recombinant MERS-CoV with nsp6 232L and 232F residues, respectively, and showed that the nsp6 L232F mutation confers higher replication competence in ex-vivo culture of human nasal and bronchial tissues and in lungs of mice experimentally infected in-vivo. Mechanistically, the nsp6 L232F mutation appeared to modulate autophagy and was associated with higher exocytic virus egress, while innate immune responses and zippering activity of the endoplasmic reticulum remained unaffected. Our study suggests that MERS-CoV nsp6 may contribute to viral adaptation to humans. This highlights the importance of continued surveillance of MERS-CoV in both camels and humans.

ISG15 driven cellular responses to virus infection.

One of the hallmarks of antiviral responses to infection is the production of interferons and subsequently of interferon stimulated genes. Interferon stimulated gene 15 (ISG15) is among the earliest and most abundant proteins induced upon interferon signalling, encompassing versatile functions in host immunity. ISG15 is a ubiquitin like modifier that can be conjugated to substrates in a process analogous to ubiquitylation and referred to as ISGylation. The free unconjugated form can either exist intracellularly or be secreted to function as a cytokine. Interestingly, ISG15 has been reported to be both advantageous and detrimental to the development of immunopathology during infection. This review describes recent findings on the role of ISG15 in antiviral responses in human infection models, with a particular emphasis on autophagy, inflammatory responses and cellular metabolism combined with viral strategies of counteracting them. The field of ISGylation has steadily gained momentum; however much of the previous studies of virus infections conducted in mouse models are in sharp contrast with recent findings in human cells, underscoring the need to summarise our current understanding of its potential antiviral function in humans and identify knowledge gaps which need to be addressed in future studies.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

Altered ISGylation drives aberrant macrophage-dependent immune responses during SARS-CoV-2 infection.

Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.

Pathogenesis and virulence of flavivirus infections.

The Flavivirus genus consists of >70 members including several that are considered significant human pathogens. Flaviviruses display a broad spectrum of diseases that can be roughly categorised into two phenotypes - systemic disease involving haemorrhage exemplified by dengue and yellow Fever virus, and neurological complications associated with the likes of West Nile and Zika viruses. Attempts to develop vaccines have been variably successful against some. Besides, mosquito-borne flaviviruses can be vertically transmitted in the arthropods, enabling long term persistence and the possibility of re-emergence. Therefore, developing strategies to combat disease is imperative even if vaccines become available. The cellular interactions of flaviviruses with their human hosts are key to establishing the viral lifecycle on the one hand, and activation of host immunity on the other. The latter should ideally eradicate infection, but often leads to immunopathological and neurological consequences. In this review, we use Dengue and Zika viruses to discuss what we have learned about the cellular and molecular determinants of the viral lifecycle and the accompanying immunopathology, while highlighting current knowledge gaps which need to be addressed in future studies.

Escaping the Lion's Den: redirecting autophagy for unconventional release and spread of viruses.

Autophagy is an evolutionarily conserved process, designed to maintain cellular homeostasis during a range of internal and external stimuli. Conventionally, autophagy is known for coordinated degradation and recycling of intracellular components and removal of cytosolic pathogens. More recently, several lines of evidence have indicated an unconventional, nondegradative role of autophagy for secretion of cargo that lacks a signal peptide. This process referred to as secretory autophagy has also been implicated in the infection cycle of several virus species. This review focuses on the current evidence available on the nondegradative features of autophagy, emphasizing its potential role and unresolved questions in the release and spread of (-) and (+) RNA viruses.

How SARS-CoV-2 (COVID-19) spreads within infected hosts - what we know so far.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19), belongs to the betacoronavirus genus and shares high homology to the severe acute respiratory syndrome coronavirus (SARS-CoV) that emerged in 2003. These are highly transmissible and pathogenic viruses which very likely originated in bats. SARS-CoV-2 uses the same receptor, angiotensin-converting enzyme 2 (ACE2) as SARS-CoV, and spreads primarily through the respiratory tract. Although several trials for vaccine development are currently underway, investigations into the virology of SARS-CoV-2 to understand the fundamental biology of the infectious cycle and the associated immunopathology underlying the clinical manifestations of COVID-19 are crucial for identification and rational design of effective therapies. This review provides an overview of how SARS-CoV-2 infects and spreads within human hosts with specific emphasis on key aspects of its lifecycle, tropism and immunopathological features.

Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles.

Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn-/- cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations - one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.

Membrane heist: Coronavirus host membrane remodeling during replication.

The ongoing pandemic of COVID-19 (Coronavirus Disease-2019), a respiratory disease caused by the novel coronavirus strain, SARS-CoV-2, has affected more than 42 million people already, with more than one million deaths worldwide (as of October 25, 2020). We are in urgent need of therapeutic interventions that target the host-virus interface, which requires a molecular understanding of the SARS-CoV-2 life-cycle. Like other positive-sense RNA viruses, coronaviruses remodel intracellular membranes to form specialized viral replication compartments, including double-membrane vesicles (DMVs), where viral RNA genome replication takes place. Here we review the current knowledge of the structure, lipid composition, function, and biogenesis of coronavirus-induced DMVs, highlighting the druggable viral and cellular factors that are involved in the formation and function of DMVs.

OTUB1 Is a Key Regulator of RIG-I-Dependent Immune Signaling and Is Targeted for Proteasomal Degradation by Influenza A NS1.

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.

Manipulation of autophagy by (+) RNA viruses.

Autophagy is an evolutionarily conserved process central to host metabolism. Among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. Besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. More recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. Interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. Particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)RNA virus infections, which benefit from the pathway without succumbing to lysosomal degradation. In this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +RNA viruses and highlight key unanswered questions in the field that we believe merit further attention.

Breaking Bad: How Viruses Subvert the Cell Cycle.

Interactions between the host and viruses during the course of their co-evolution have not only shaped cellular function and the immune system, but also the counter measures employed by viruses. Relatively small genomes and high replication rates allow viruses to accumulate mutations and continuously present the host with new challenges. It is therefore, no surprise that they either escape detection or modulate host physiology, often by redirecting normal cellular pathways to their own advantage. Viruses utilize a diverse array of strategies and molecular targets to subvert host cellular processes, while evading detection. These include cell-cycle regulation, major histocompatibility complex-restricted antigen presentation, intracellular protein transport, apoptosis, cytokine-mediated signaling, and humoral immune responses. Moreover, viruses routinely manipulate the host cell cycle to create a favorable environment for replication, largely by deregulating cell cycle checkpoints. This review focuses on our current understanding of the molecular aspects of cell cycle regulation that are often targeted by viruses. Further study of their interactions should provide fundamental insights into cell cycle regulation and improve our ability to exploit these viruses.

Perturbation of Intracellular Cholesterol and Fatty Acid Homeostasis During Flavivirus Infections.

Cellular lipid homeostasis is maintained through an intricately linked array of anabolic and catabolic pathways. Upon flavivirus infections, these are significantly altered: on the one hand, these viruses can co-opt lipid metabolic pathways to generate ATP to facilitate replication, or to synthesize membrane components to generate replication sites; on the other hand, more recent evidence suggests counter strategies employed by host cells, which actively modulate several of these networks in response to infection, enhancing interferon signaling by doing so, and thus creating an antiviral environment. In this review, we discuss recent data on mechanisms of alteration of lipid metabolic pathways during infection by flaviviruses, with a focus on cholesterol and fatty acid biosynthesis, which can be manipulated by the invading viruses to support replication, but can also be modulated by the host immune system itself, as a means to fight infection.