SnapShot: antiviral restriction factors.

Restriction factors are cellular proteins that inhibit viruses at different steps of their replication cycle and represent an important first line of defense against viral pathogens. This SnapShot provides an overview of cell-intrinsic antiviral factors, describes their properties, and illustrates the striking variety of antiviral mechanisms as well the sophisticated viral countermeasures. To view this SnapShot, open or download the PDF.

Not all viruses cause disease: HERV-K(HML-2) in healthy human tissues.

Human endogenous retroviruses (HERVs) make up a significant part of our genome. While their expression is frequently associated with disease, a new study in PLOS Biology found that HERV-K(HML-2) is expressed in more than 50 healthy tissues.

ORF3c is expressed in SARS-CoV-2-infected cells and inhibits innate sensing by targeting MAVS.

Most SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While the majority of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. One example is the ORF3a sgRNA that also encodes ORF3c, an enigmatic 41-amino-acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2-infected cells and suppresses RIG-I- and MDA5-mediated IFN-ß induction. ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage, and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c, demonstrating that this reading frame is not essential for efficient viral replication in vivo and is likely compensated by other viral proteins. In agreement with this, disruption of ORF3c does not significantly affect SARS-CoV-2 replication in CaCo-2, CaLu-3, or Rhinolophus alcyone cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

Tetherin: holding on and letting go.

Mammalian cells are equipped with so-called "restriction factors" that suppress virus replication and help to prevent virus transmission from one species to another. This Essay discusses the host restriction factor tetherin, which blocks the release of enveloped viruses like HIV-1, and the factors evolved by primate lentiviruses, such as Vpu and Nef, that antagonize tetherin's action.

Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

HIV-1 Nef-mediated downregulation of CD155 results in viral restriction by KIR2DL5+ NK cells.

Antiviral NK cell activity is regulated through the interaction of activating and inhibitory NK cell receptors with their ligands on infected cells. HLA class I molecules serve as ligands for most killer cell immunoglobulin-like receptors (KIRs), but no HLA class I ligands for the inhibitory NK cell receptor KIR2DL5 have been identified to date. Using a NK cell receptor/ligand screening approach, we observed no strong binding of KIR2DL5 to HLA class I or class II molecules, but confirmed that KIR2DL5 binds to the poliovirus receptor (PVR, CD155). Functional studies using primary human NK cells revealed a significantly decreased degranulation of KIR2DL5+ NK cells in response to CD155-expressing target cells. We subsequently investigated the role of KIR2DL5/CD155 interactions in HIV-1 infection, and showed that multiple HIV-1 strains significantly decreased CD155 expression levels on HIV-1-infected primary human CD4+ T cells via a Nef-dependent mechanism. Co-culture of NK cells with HIV-1-infected CD4+ T cells revealed enhanced anti-viral activity of KIR2DL5+ NK cells against wild-type versus Nef-deficient viruses, indicating that HIV-1-mediated downregulation of CD155 renders infected cells more susceptible to recognition by KIR2DL5+ NK cells. These data show that CD155 suppresses the antiviral activity of KIR2DL5+ NK cells and is downmodulated by HIV-1 Nef protein as potential trade-off counteracting activating NK cell ligands, demonstrating the ability of NK cells to counteract immune escape mechanisms employed by HIV-1.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Less is more: Biased loss of CpG dinucleotides strengthens antiviral immunity.

Viruses may not only affect our daily lives but also shape our genome evolution. A recent study shows that the zinc-finger antiviral protein (ZAP) drives CpG suppression in a biased manner. Genes involved in the defense against viral invaders are particularly CpG suppressed to avoid self-targeting and to promote an effective immune response.

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins.

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection.

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.

Phase retrieval for the generation of arbitrary intensity distributions using an optofluidic phase shifter.

An optofluidic phase shifter can be used to generate virtually arbitrary intensity patterns, but only if the phase shift generated by the controllably deformed fluidic surface can be appropriately defined. To enable this functionality, we present two phase retrieval algorithms based on neural networks and least-squares optimization which are used to determine the necessary phase profile to generate a desired target intensity pattern with high accuracy. We demonstrate the utility of the algorithms by showing experimentally the ability of an optofluidic phase shifter to generate arbitrary complex intensity distributions.

Switching Sides: How Endogenous Retroviruses Protect Us from Viral Infections.

Long disregarded as junk DNA or genomic dark matter, endogenous retroviruses (ERVs) have turned out to represent important components of the antiviral immune response. These remnants of once-infectious retroviruses not only regulate cellular immune activation, but may even directly target invading viral pathogens. In this Gem, we summarize mechanisms by which retroviral fossils protect us from viral infections. One focus will be on recent advances in the role of ERVs as regulators of antiviral gene expression.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

HIV-1 infection activates endogenous retroviral promoters regulating antiviral gene expression.

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.

Validating Automatic Concept-Based Explanations for AI-Based Digital Histopathology.

Digital histopathology poses several challenges such as label noise, class imbalance, limited availability of labelled data, and several latent biases to deep learning, negatively influencing transparency, reproducibility, and classification performance. In particular, biases are well known to cause poor generalization. Proposed tools from explainable artificial intelligence (XAI), bias detection, and bias discovery suffer from technical challenges, complexity, unintuitive usage, inherent biases, or a semantic gap. A promising XAI method, not studied in the context of digital histopathology is automated concept-based explanation (ACE). It automatically extracts visual concepts from image data. Our objective is to evaluate ACE's technical validity following design science principals and to compare it to Guided Gradient-weighted Class Activation Mapping (Grad-CAM), a conventional pixel-wise explanation method. To that extent, we created and studied five convolutional neural networks (CNNs) in four different skin cancer settings. Our results demonstrate that ACE is a valid tool for gaining insights into the decision process of histopathological CNNs that can go beyond explanations from the control method. ACE validly visualized a class sampling ratio bias, measurement bias, sampling bias, and class-correlated bias. Furthermore, the complementary use with Guided Grad-CAM offers several benefits. Finally, we propose practical solutions for several technical challenges. In contradiction to results from the literature, we noticed lower intuitiveness in some dermatopathology scenarios as compared to concept-based explanations on real-world images.

Tunable fluidic lens with a dynamic high-order aberration control.

Fluidic lenses based on electrowetting actuation are attractive for their wide focal tuning range, yet are limited by optical aberrations, either intrinsic to the lenses themselves or due to the optical imaging systems in which they are employed. However, the ability to control the meniscus shape that forms the lens refractive surface with a high degree of spatial accuracy will allow correction of and compensation for a wide range of these aberrations. We demonstrate here for what we believe, to the best of our knowledge, is the first time a tunable optofluidic lens controlled by 32 azimuthally placed electrodes for which most aberrations up to the fourth radial Zernike order may be corrected. Using both wavefront sensing and sensorless wavefront estimation techniques, it is shown that focal length tunability with a significant reduction in imaging aberrations and the ability to compensate for externally induced aberrations may be achieved using a single component.

Primary HIV-1 Strains Use Nef To Downmodulate HLA-E Surface Expression.

Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surfaces of infected cells. Recent evidence identified HLA-E, a nonclassical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8+ T-cell and natural killer (NK) cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1-infected cells. Here, we examined HLA-E surface levels following infection of CD4+ T cells with primary HIV-1 strains and observed that a subset downregulated HLA-E. Two primary strains of HIV-1 that induced the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in a T-cell line, as well as tail swap experiments exchanging the cytoplasmic tail of HLA-A2 with that of HLA-E, demonstrated that Nef modulated HLA-E surface levels and targeted the cytoplasmic tail of HLA-E. Furthermore, infection of primary CD4+ T cells with HIV-1 mutants showed that a lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, the results of this study demonstrate for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4+ T cells, potentially rendering them less vulnerable to CD8+ T-cell recognition but at increased risk of NKG2A+ NK cell killing.IMPORTANCE For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic-T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that HIV-1 infection thereby maintained inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell line-adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as an important modulator of CD8+ T-cell and NKG2A+ NK cell functions, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.