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Innate immune sensors and restriction factors are cellular proteins that synergize to build an effective first line of defense against viral infections. Innate sensors are usually constitutively expressed and capable of detecting pathogen-associated molecular patterns (PAMPs) via specific pattern recognition receptors (PRRs) to stimulate the immune response. Restriction factors are frequently upregulated by interferons (IFNs) and may inhibit viral pathogens at essentially any stage of their replication cycle. Members of the Pyrin and hematopoietic interferon-inducible nuclear (HIN) domain (PYHIN) family have initially been recognized as important sensors of foreign nucleic acids and activators of the inflammasome and the IFN response. Accumulating evidence shows, however, that at least three of the four members of the human PYHIN family restrict viral pathogens independently of viral sensing and innate immune activation. In this review, we provide an overview on the role of human PYHIN proteins in the innate antiviral immune defense and on viral countermeasures.

Restriction factors are structurally and functionally diverse cellular proteins that constitute a first line of defense against viral pathogens. Exceptions exist, but typically these proteins are upregulated by interferons (IFNs), target viral components, and are rapidly evolving due to the continuous virus–host arms race. Restriction factors may target HIV replication at essentially each step of the retroviral replication cycle, and the suppression of viral transcription and the degradation of viral RNA transcripts are emerging as major innate immune defense mechanisms. Recent data show that some antiviral factors, such as the tripartite motif-containing protein 22 (TRIM22) and the γ-IFN-inducible protein 16 (IFI16), do not target HIV-1 itself but limit the availability of the cellular transcription factor specificity protein 1 (Sp1), which is critical for effective viral gene expression. In addition, several RNA-interacting cellular factors including RNAse L, the NEDD4-binding protein 1 (N4BP1), and the zinc finger antiviral protein (ZAP) have been identified as important immune effectors against HIV-1 that may be involved in the maintenance of the latent viral reservoirs, representing the major obstacle against viral elimination and cure. Here, we review recent findings on specific cellular antiviral factors targeting HIV-1 transcription or viral RNA transcripts and discuss their potential role in viral latency.

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.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

Replication of human pathogenic viruses such as HIV-1 is constantly challenged by selective pressures exerted by a large arsenal of host sensors and restriction factors. Consecutively, this drives selection of viruses able to neutralize or evade such host defenses. Human PYHIN proteins (AIM2, IFI16, MNDA and IFIX) are emerging players of the cell-intrinsic immune response. While AIM2 is a well-established cytosolic sensor of exogenous DNA, the role of IFI16 as a nucleic acid sensor is rather controversial. In turn, IFI16 is mostly known to inhibit viral replication by silencing viral gene expression of several DNA viruses. IFIX and MNDA have been shown to be antivirally active against two different herpesviruses, but their precise mechanism remained elusive. As such, the goal of my thesis was to elucidate if and how PYHIN proteins restrict replication of primary HIV-1 isolates. My findings revealed that IFI16, IFIX, MNDA and AIM2 are readily detectable in HIV-1 primary target cells and that all but AIM2 restrict viral transcription. Mechanistic studies showed that nuclear PYHIN proteins inhibit lentiviral gene expression by binding to and reducing the availability of the host transcription factor Sp1. In contrast, AIM2 did not exert antiviral activity. This is due to its physiological cytosolic compartmentalization, as artificial relocalization into the nucleus rendered AIM2 antivirally active. Deletion mutagenesis and functional analyses further revealed that the N-terminal Pyrin domain (PYD) is sufficient for the antiviral function, suggesting that the antiviral mechanism is independent of the DNA interaction and sensing functions of PYHIN proteins. Further experiments in both primary CD4+ T and in a latently infected T cell line further revealed the crucial role of Sp1 in promoting viral reactivation from latency and how this process is antagonized by IFI16. Interestingly, highly prevalent subtype C viruses evade these restriction factors by reducing their dependency on Sp1 for efficient transcription, and this effect seems to be dependent on the proviral LTRs.In conclusion, my results illustrate how nuclear PYHIN proteins commonly inhibit HIV-1 replication by targeting the host transcription factor Sp1. Finding that a highly prevalent HIV-1 subtype efficiently evades these restriction factors should stimulate further studies aimed at understanding which region in the proviral LTRs is responsible for this effect as well as the underlying mechanism. Notably, Sp1 is required for basal and induced expression of a plethora of cellular genes and it is known to play an important role in cancer. Hence, the PYHIN-Sp1 interaction should be additionally investigated outside of the context of viral infection, to shed more light on the physiological role of PYHIN proteins.

Modeling real-world systems requires accounting for noise - whether it arises from unpredictable fluctuations in financial markets, irregular rhythms in biological systems, or environmental variability in ecosystems. While the behavior of such systems can often be described by stochastic differential equations, a central challenge is understanding how noise influences the inference of system parameters and dynamics from data. Traditional symbolic regression methods can uncover governing equations but typically ignore uncertainty. Conversely, Gaussian processes provide principled uncertainty quantification but offer little insight into the underlying dynamics. In this work, we bridge this gap with a hybrid symbolic regression-probabilistic machine learning framework that recovers the symbolic form of the governing equations while simultaneously inferring uncertainty in the system parameters. The framework combines deep symbolic regression with Gaussian process-based maximum likelihood estimation to separately model the deterministic dynamics and the noise structure, without requiring prior assumptions about their functional forms. We verify the approach on numerical benchmarks, including harmonic, Duffing, and van der Pol oscillators, and validate it on an experimental system of coupled biological oscillators exhibiting synchronization, where the algorithm successfully identifies both the symbolic and stochastic components. The framework is data-efficient, requiring as few as 100-1000 data points, and robust to noise - demonstrating its broad potential in domains where uncertainty is intrinsic and both the structure and variability of dynamical systems must be understood.

Viruses manipulate the host cell membrane of infected cells for evasion of antiviral immunity, prevention of superinfection and optimization of viral replication and spread. The Ebola virus glycoprotein (EBOV GP) mediates virus entry, but is also known as important factor for subversion of the hosts antiviral immune response. We characterized the dysregulation of cell surface-residing proteins by EBOV GP and found that among several membrane proteins GP interferes with the tetraspanins CD81, CD63 and CD9. This was a conserved function of several filoviral GPs and not observable for viral glycoproteins of other virus families. While CD63 and CD9 were largely dispensable for EBOV replication, CD81 suppressed virus-like particle entry and replication at multiple steps. This phenotype might be explainable by sustained suppression of NFκB by CD81, that is otherwise activated by VP40 and EBOV trVLP replication. We further demonstrate that not only GP but also VP40 interferes with CD81 functionality and that antibody-mediated clustering of CD81 suppresses EBOV infection. Altogether, the tetraspanin CD81 emerges as druggable NFκB and EBOV-inhibiting factor, supporting an important role of NFκB in EBOV replication and potentially virus-induced immunopathogenesis.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredDeutsche Forschungsgemeinschaft, SCHI 1073/10-1, 399732171