A Variety of Mouse PYHIN Proteins Restrict Murine and Human Retroviruses.

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.

Increased in vitro Anti-HIV Activity of Caffeinium-Functionalized Polyoxometalates.

Polyoxometalates (POMs), molecular metal oxide anions, are inorganic clusters with promising antiviral activity. Herein we report increased anti-HIV-1 activity of a POM when electrostatically combined with organic counter-cations. To this end, Keggin-type cerium tungstate POMs have been combined with organic methyl-caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and mode of action have been studied. The novel compound, Caf4 K[ß2 -CeSiW11 O39 ]×H2 O, exhibits sub-nanomolar antiviral activity and inhibits HIV-1 infectivity by acting on an early step of the viral infection cycle. This work demonstrates that combination of POM anions and organic bioactive cations can be a powerful new strategy to increase antiviral activity of these inorganic compounds.

IFI16 knockdown in primary HIV-1 target cells.

IFI16 is an important player of the host intrinsic immune response. Among others, it has been reported to sense intermediate products of HIV-1 reverse transcription in the cytosol and to sequester the transcription factor Sp1 in the nucleus to attenuate viral gene expression. Here, we present three different methods to reduce IFI16 protein expression levels in HIV-1 primary target cells. These techniques can be adapted for the investigation of other cellular factors in primary macrophages and CD4+ T lymphocytes. For complete details on the use and execution of this protocol, please refer to Hotter et al. (2019) and Bosso et al. (2020).

Emerging Role of PYHIN Proteins as Antiviral Restriction Factors.

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.

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.

Exploiting the human peptidome for novel antimicrobial and anticancer agents.

Infectious diseases and cancers are leading causes of death and pose major challenges to public health. The human peptidome encompasses millions of compounds that display an enormous structural and functional diversity and represents an excellent source for the discovery of endogenous agents with antimicrobial and/or anticancer activity. Here, we discuss how to exploit the human peptidome for novel antimicrobial and anticancer agents through the generation of peptide libraries from human body fluids and tissues and stepwise purification of bioactive compounds.

Human Cytomegalovirus Particles Treated with Specific Antibodies Induce Intrinsic and Adaptive but Not Innate Immune Responses.

Human cytomegalovirus (HCMV) persistently infects 40% to 100% of the human population worldwide. Experimental and clinical evidence indicates that humoral immunity to HCMV plays an important role in restricting virus dissemination and protecting the infected host from disease. Specific immunoglobulin preparations from pooled plasma of adults selected for high titers of HCMV antibodies have been used for the prevention of CMV disease in transplant recipients and pregnant women. Even though incubation of HCMV particles with these preparations leads to the neutralization of viral infectivity, it is still unclear whether the antibody-treated HCMV particles (referred to here as HCMV-Ab) enter the cells and modulate antiviral immune responses. Here we demonstrate that HCMV-Ab did enter macrophages. HCMV-Ab did not initiate the expression of immediate early antigens (IEAs) in macrophages, but they induced an antiviral state and rendered the cells less susceptible to HCMV infection upon challenge. Resistance to HCMV infection seemed to be due to the activation of intrinsic restriction factors and was independent of interferons. In contrast to actively infected cells, autologous NK cells did not degranulate against HCMV-Ab-treated macrophages, suggesting that these cells may not be eliminated by innate effector cells. Interestingly, HCMV-Ab-treated macrophages stimulated the proliferation of autologous adaptive CD4+ and CD8+ T cells. Our findings not only expand the current knowledge on virus-antibody immunity but may also be relevant for future vaccination strategies.IMPORTANCE Human cytomegalovirus (HCMV), a common herpesvirus, establishes benign but persistent infections in immunocompetent hosts. However, in subjects with an immature or dysfunctional immune system, HCMV is a major cause of morbidity and mortality. Passive immunization has been used in different clinical settings with variable clinical results. Intravenous hyperimmune globulin preparations (IVIg) are obtained from pooled adult human plasma selected for high anti-CMV antibody titers. While HCMV neutralization can be shown in vitro using different systems, data are lacking regarding the cross-influence of IVIg administration on the cellular immune responses. The aim of this study was to evaluate the effects of IVIg on distinct components of the immune response against HCMV, including antigen presentation by macrophages, degranulation of innate natural killer cells, and proliferation of adaptive CD4+ and CD8+ T cells.