Functional-genomic analysis reveals intraspecies diversification of antiviral receptor transporter proteins in Xenopus laevis.

The Receptor Transporter Protein (RTP) family is present in most, if not all jawed vertebrates. Most of our knowledge of this protein family comes from studies on mammalian RTPs, which are multi-function proteins that regulate cell-surface G-protein coupled receptor levels, influence olfactory system development, regulate immune signaling, and directly inhibit viral infection. However, mammals comprise less than one-tenth of extant vertebrate species, and our knowledge about the expression, function, and evolution of non-mammalian RTPs is limited. Here, we explore the evolutionary history of RTPs in vertebrates. We identify signatures of positive selection in many vertebrate RTP clades and characterize multiple, independent expansions of the RTP family outside of what has been described in mammals. We find a striking expansion of RTPs in the African clawed frog, Xenopus laevis, with 11 RTPs in this species as opposed to 1 to 4 in most other species. RNA sequencing revealed that most X. laevis RTPs are upregulated following immune stimulation. In functional assays, we demonstrate that at least three of these X. laevis RTPs inhibit infection by RNA viruses, suggesting that RTP homologs may serve as antiviral effectors outside of Mammalia.

Shiftless inhibits flavivirus replication in vitro and is neuroprotective in a mouse model of Zika virus pathogenesis.

Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl-/- knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.

IFIT1 is rapidly evolving and exhibits disparate antiviral activities across 11 mammalian orders.

Mammalian mRNAs possess an N7-methylguanosine (m7G) cap and 2'O methylation of the initiating nucleotide at their 5' end, whereas certain viral RNAs lack these characteristic features. The human antiviral restriction factor IFIT1 recognizes and binds to specific viral RNAs that lack the 5' features of host mRNAs, resulting in targeted suppression of viral RNA translation. This interaction imposes significant host-driven evolutionary pressures on viruses, and many viruses have evolved mechanisms to evade the antiviral action of human IFIT1. However, little is known about the virus-driven pressures that may have shaped the antiviral activity of IFIT1 genes across mammals. Here, we take an evolution-guided approach to show that the IFIT1 gene is rapidly evolving in multiple mammalian clades, with positive selection acting upon several residues in distinct regions of the protein. In functional assays with 39 IFIT1s spanning diverse mammals, we demonstrate that IFIT1 exhibits a range of antiviral phenotypes, with many orthologs lacking antiviral activity against viruses that are strongly suppressed by other IFIT1s. We further show that IFIT1s from human and a bat, the black flying fox, inhibit Venezuelan equine encephalitis virus (VEEV) and strongly bind to Cap0 RNAs. Unexpectedly, chimpanzee IFIT1, which differs from human IFIT1 by only 8 amino acids, does not inhibit VEEV infection and exhibits minimal Cap0 RNA-binding. In mutagenesis studies, we determine that amino acids 364 and 366, with the latter undergoing positive selection, are sufficient to confer the differential anti-VEEV activity between human and chimpanzee IFIT1. These data suggest that virus-host genetic conflicts have influenced the antiviral specificity of IFIT1 across diverse mammalian orders.

Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo.

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spikes from multiple SARS-CoV-2 variants and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for the delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrated protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses. IMPORTANCE: The rapid evolution of SARS-CoV-2 variants poses a challenge for immune recognition and antibody therapies. However, the virus is constrained by the requirement that it recognizes a human host receptor protein. A recombinant ACE2 could protect against SARS-CoV-2 infection by functioning as a soluble decoy receptor. We designed a mutant version of ACE2 with impaired catalytic activity to enable the purification of the protein using a single affinity purification step. This protein can be nebulized and retains the ability to bind the relevant domains from SARS-CoV-1 and SARS-CoV-2. Moreover, this protein inhibits viral infection against a panel of coronaviruses in cells. Finally, we developed an aerosolized delivery system for animal studies and show the modified ACE2 offers protection in an animal model of COVID-19. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2.

Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints.

Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences.

Structural homology screens reveal poxvirus-encoded proteins impacting inflammasome-mediated defenses.

Viruses acquire host genes via horizontal gene transfer and can express them to manipulate host biology during infections. Some viral and host homologs retain sequence identity, but evolutionary divergence can obscure host origins. We used structural modeling to compare vaccinia virus proteins with metazoan proteomes. We identified vaccinia A47L as a homolog of gasdermins, the executioners of pyroptosis. An X-ray crystal structure of A47 confirmed this homology and cell-based assays revealed that A47 inhibits pyroptosis. We also identified vaccinia C1L as the product of a cryptic gene fusion event coupling a Bcl-2 related fold with a pyrin domain. C1 associates with components of the inflammasome, a cytosolic innate immune sensor involved in pyroptosis, yet paradoxically enhances inflammasome activity, suggesting a benefit to poxvirus replication in some circumstances. Our findings demonstrate the potential of structural homology screens to reveal genes that viruses capture from hosts and repurpose to benefit viral fitness.

Structural homology screens reveal host-derived poxvirus protein families impacting inflammasome activity.

Viruses acquire host genes via horizontal transfer and can express them to manipulate host biology during infections. Some homologs retain sequence identity, but evolutionary divergence can obscure host origins. We use structural modeling to compare vaccinia virus proteins with metazoan proteomes. We identify vaccinia A47L as a homolog of gasdermins, the executioners of pyroptosis. An X-ray crystal structure of A47 confirms this homology, and cell-based assays reveal that A47 interferes with caspase function. We also identify vaccinia C1L as the product of a cryptic gene fusion event coupling a Bcl-2-related fold with a pyrin domain. C1 associates with components of the inflammasome, a cytosolic innate immune sensor involved in pyroptosis, yet paradoxically enhances inflammasome activity, suggesting differential modulation during infections. Our findings demonstrate the increasing power of structural homology screens to reveal proteins with unique combinations of domains that viruses capture from host genes and combine in unique ways.

Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo.

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spike from multiple SARS-CoV-2 variants, and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrate protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.

FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms.

The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.

When viruses do not go gentle into that good night.

A recent paper in Cell reports the discovery of a receptor for simian hemorrhagic fever virus and suggests that it may be poised to spill over into humans. This study highlights the importance of devoting resources to currently obscure animal viruses that may pose a threat to human health.

Evolution of the interferon response: lessons from ISGs of diverse mammals.

The vertebrate interferon (IFN) response controls viral infections by inducing hundreds of interferon-stimulated genes (ISGs), many of which encode 'restriction factors' that uniquely target certain viruses. ISG studies have historically had a human-centric focus, which is justified because these natural defense mechanisms might be leveraged to treat human viral disease. However, certain mammals are reservoirs for zoonotic viruses that can 'spill over' into humans. Additionally, restriction factors have prominent roles in the ongoing evolutionary genetic conflicts between viruses and their hosts. Thus, there is a growing need to understand antiviral IFN/ISG responses in other species, particularly in known reservoirs of zoonotic viruses. This review focuses on functional and evolutionary insight into antiviral IFN responses that have been obtained from studying non-model mammalian species.

RTP4 Is a Potent IFN-Inducible Anti-flavivirus Effector Engaged in a Host-Virus Arms Race in Bats and Other Mammals.

Among mammals, bats are particularly rich in zoonotic viruses, including flaviviruses. Certain bat species can be productively yet asymptomatically infected with viruses that cause overt disease in other species. However, little is known about the antiviral effector repertoire in bats relative to other mammals. Here, we report the black flying fox receptor transporter protein 4 (RTP4) as a potent interferon (IFN)-inducible inhibitor of human pathogens in the Flaviviridae family, including Zika, West Nile, and hepatitis C viruses. Mechanistically, RTP4 associates with the flavivirus replicase, binds viral RNA, and suppresses viral genome amplification. Comparative approaches revealed that RTP4 undergoes positive selection, that a flavivirus can mutate to escape RTP4-imposed restriction, and that diverse mammalian RTP4 orthologs exhibit striking patterns of specificity against distinct Flaviviridae members. Our findings reveal an antiviral mechanism that has likely adapted over 100 million years of mammalian evolution to accommodate unique host-virus genetic conflicts.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.

LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step.

Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of interferon-stimulated genes (ISGs). In a screen to identify antiviral ISGs, we unexpectedly found that LY6E, a member of the LY6/uPAR family, enhanced viral infection. Here, we show that viral enhancement by ectopically expressed LY6E extends to several cellular backgrounds and affects multiple RNA viruses. LY6E does not impair IFN antiviral activity or signaling, but rather promotes viral entry. Using influenza A virus as a model, we narrow the enhancing effect of LY6E to uncoating after endosomal escape. Diverse mammalian orthologs of LY6E also enhance viral infectivity, indicating evolutionary conservation of function. By structure-function analyses, we identify a single amino acid in a predicted loop region that is essential for viral enhancement. Our study suggests that LY6E belongs to a class of IFN-inducible host factors that enhance viral infectivity without suppressing IFN antiviral activity.

A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication.

The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.