Host-specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.

Recurrent Loss of Macrodomain Activity in Host Immunity and Viral Proteins.

Protein post-translational modifications (PTMs) are an important battleground in the evolutionary arms races that are waged between the host innate immune system and viruses. One such PTM, ADP-ribosylation, has recently emerged as an important mediator of host antiviral immunity. Important for the host-virus conflict over this PTM is the addition of ADP-ribose by PARP proteins and removal of ADP-ribose by macrodomain-containing proteins. Interestingly, several host proteins, known as macroPARPs, contain macrodomains as well as a PARP domain, and these proteins are both important for the host antiviral immune response and evolving under very strong positive (diversifying) evolutionary selection. In addition, several viruses, including alphaviruses and coronaviruses, encode one or more macrodomains. Despite the presence of the conserved macrodomain fold, the enzymatic activity of many of these proteins has not been characterized. Here, we perform evolutionary and functional analyses to characterize the activity of macroPARP and viral macrodomains. We trace the evolutionary history of macroPARPs in metazoans and show that PARP9 and PARP14 contain a single active macrodomain, whereas PARP15 contains none. Interestingly, we also reveal several independent losses of macrodomain enzymatic activity within mammalian PARP14, including in the bat, ungulate, and carnivore lineages. Similar to macroPARPs, coronaviruses contain up to three macrodomains, with only the first displaying catalytic activity. Intriguingly, we also reveal the recurrent loss of macrodomain activity within the alphavirus group of viruses, including enzymatic loss in insect-specific alphaviruses as well as independent enzymatic losses in two human-infecting viruses. Together, our evolutionary and functional data reveal an unexpected turnover in macrodomain activity in both host antiviral proteins and viral proteins.

Tripping the wire: sensing of viral protease activity by CARD8 and NLRP1 inflammasomes.

Host innate immune sensors are vital for the initial detection of pathogen infection. Such sensors thus need to constantly adapt in escalating evolutionary arms races with pathogens. Recently, two inflammasome-forming proteins, CARD8 and NLRP1, have emerged as innate immune sensors for the enzymatic activity of virus-encoded proteases. When cleaved within a rapidly evolving 'tripwire' region, CARD8 and NLRP1 assemble into inflammasomes that initiate pyroptotic cell death and pro-inflammatory cytokine release as a form of effector-triggered immunity. Short motifs in the CARD8 and NLRP1 tripwires mimic the protease-specific cleavage sites of picornaviruses, coronaviruses, and HIV-1, providing virus-specific sensing that can rapidly change between closely related hosts and within the human population. Recent work highlights the evolutionary arms races between viral proteases and NLRP1 and CARD8, including insights into the mechanisms of inflammasome activation, host diversity of viral sensing, and means that viruses have evolved to avoid tripping the wire.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Antiviral function and viral antagonism of the rapidly evolving dynein activating adaptor NINL.

Viruses interact with the intracellular transport machinery to promote viral replication. Such host-virus interactions can drive host gene adaptation, leaving signatures of pathogen-driven evolution in host genomes. Here, we leverage these genetic signatures to identify the dynein activating adaptor, ninein-like (NINL), as a critical component in the antiviral innate immune response and as a target of viral antagonism. Unique among genes encoding components of active dynein complexes, NINL has evolved under recurrent positive (diversifying) selection, particularly in its carboxy-terminal cargo-binding region. Consistent with a role for NINL in host immunity, we demonstrate that NINL knockout cells exhibit an impaired response to interferon, resulting in increased permissiveness to viral replication. Moreover, we show that proteases encoded by diverse picornaviruses and coronaviruses cleave and disrupt NINL function in a host- and virus-specific manner. Our work reveals the importance of NINL in the antiviral response and the utility of using signatures of host-virus genetic conflicts to uncover new components of antiviral immunity and targets of viral antagonism.

Humans and viruses are locked in an evolutionary arms race. Viruses hijack cells, using their resources and proteins to build more viral particles; the cells fight back, calling in the immune system to fend off the attack. Both actors must constantly and quickly evolve to keep up with each other. This genetic conflict has been happening for millions of years, and the indelible marks it has left on genes can serve to uncover exactly how viruses interact with the organisms they invade. One hotspot in this host-virus conflict is the complex network of molecules that help to move cargo inside a cell. This system transports elements of the immune system, but viruses can also harness it to make more of themselves. Scientists still know very little about how viruses and the intracellular transport machinery interact, and how this impacts viral replication and the immune response. Stevens et al. therefore set out to identify new interactions between viruses and the transport system by using clues left in host genomes by evolution. They focused on dynein, a core component of this machinery which helps to haul molecular actors across a cell. To do so, dynein relies on adaptor molecules such as 'Ninein-like', or NINL for short. Closely examining the gene sequence for NINL across primates highlighted an evolutionary signature characteristic of host-virus genetic conflicts; this suggests that the protein may be used by viruses to reproduce, or by cells to fend off infection. And indeed, human cells lacking the NINL gene were less able to defend themselves, allowing viruses to grow much faster than normal. Further work showed that NINL was important for a major type of antiviral immune response. As a potential means to sabotage this defence mechanism, some viruses cleave NINL at specific sites and disrupt its role in intracellular transport. Better antiviral treatments are needed to help humanity resist old foes and new threats alike. The work by Stevens et al. demonstrates how the information contained in host genomes can be leveraged to understand what drives susceptibility to an infection, and to pinpoint molecular actors which could become therapeutic targets.

Running With Scissors: Evolutionary Conflicts Between Viral Proteases and the Host Immune System.

Many pathogens encode proteases that serve to antagonize the host immune system. In particular, viruses with a positive-sense single-stranded RNA genome [(+)ssRNA], including picornaviruses, flaviviruses, and coronaviruses, encode proteases that are not only required for processing viral polyproteins into functional units but also manipulate crucial host cellular processes through their proteolytic activity. Because these proteases must cleave numerous polyprotein sites as well as diverse host targets, evolution of these viral proteases is expected to be highly constrained. However, despite this strong evolutionary constraint, mounting evidence suggests that viral proteases such as picornavirus 3C, flavivirus NS3, and coronavirus 3CL, are engaged in molecular 'arms races' with their targeted host factors, resulting in host- and virus-specific determinants of protease cleavage. In cases where protease-mediated cleavage results in host immune inactivation, recurrent host gene evolution can result in avoidance of cleavage by viral proteases. In other cases, such as recently described examples in NLRP1 and CARD8, hosts have evolved 'tripwire' sequences that mimic protease cleavage sites and activate an immune response upon cleavage. In both cases, host evolution may be responsible for driving viral protease evolution, helping explain why viral proteases and polyprotein sites are divergent among related viruses despite such strong evolutionary constraint. Importantly, these evolutionary conflicts result in diverse protease-host interactions even within closely related host and viral species, thereby contributing to host range, zoonotic potential, and pathogenicity of viral infection. Such examples highlight the importance of examining viral protease-host interactions through an evolutionary lens.