Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

SAMHD1 is a host triphosphohydrolase that degrades intracellular deoxynucleoside triphosphates (dNTPs) to a lower level that restricts viral DNA synthesis, and thus prevents replication of diverse viruses in non-dividing cells. Recent progress indicates that SAMHD1 negatively regulates antiviral innate immune responses and inflammation through interacting with various key proteins in immune signaling and DNA damage repair pathways. SAMHD1 can also modulate antibody production in adaptive immune responses. In this review, we summarize how SAMHD1 regulates antiviral immune responses through distinct mechanisms and discuss the implications of these new functions of SAMHD1. Furthermore, we propose important new questions and future directions that can advance functional and mechanistic studies of SAMHD1-mediated immune regulation during viral infections.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-β, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus–infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-β mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2 NS1217 ) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2 NS1230 and H9N2 NS1237 ). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madin-Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2 NS1230 and H9N2 NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2 NS1230 and H9N2 NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.

Given the present extensive co-circulation in pigs of Eurasian avian-like (EA) swine H1N1 and 2009 pandemic (pdm/09) H1N1 viruses, reassortment between them is highly plausible but largely uncharacterized. Here, experimentally co-infected pigs with a representative EA virus and a pdm/09 virus yielded 55 novel reassortant viruses that could be categorized into 17 genotypes from Gt1 to Gt17 based on segment segregation. Majority of novel reassortants were isolated from the lower respiratory tract. Most of reassortant viruses were more pathogenic and contagious than the parental EA viruses in mice and guinea pigs. The most transmissible reassortant genotypes demonstrated in guinea pigs (Gt2, Gt3, Gt7, Gt10 and Gt13) were also the most lethal in mice. Notably, nearly all these highly virulent reassortants (all except Gt13) were characterized with possession of EA H1 and full complement of pdm/09 ribonucleoprotein genes. Compositionally, we demonstrated that EA H1-222G contributed to virulence by its ability to bind avian-type sialic acid receptors, and that pdm/09 RNP conferred the most robust polymerase activity to reassortants. The present study revealed high reassortment compatibility between EA and pdm/09 viruses in pigs, which could give rise to progeny reassortant viruses with enhanced virulence and transmissibility in mice and guinea pig models.

Bacterial keratitis in animals presents challenges due to ocular structural barriers, hindering effective drug delivery. In this study, we used biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) to encapsulate the naturally occurring antimicrobial peptide OH-CATH30, an alternative to conventional antibiotics, for the treatment of bacterial keratitis in animals. Microspheres (MS) were prepared using a modified water-in-oil-in-water (W/O/W) double-emulsion method with optimized osmotic pressure. We conducted comprehensive evaluations, including in vitro characterization, encapsulation efficiency determination, in vitro release kinetics, and in vivo/vitro assessments of irritation and bacterial inhibition. The optimized method yielded microspheres with impressive encapsulation efficiency of 75.2 ± 3.62% and a loading capacity of 18.25 ± 5.73%, exhibiting a well-defined particle size distribution (200–1000 nm) and a ζ-potential of −17.3 ± 1.91 mV. The microspheres demonstrated initial burst release followed by sustained and controlled release in vitro. Both in vitro and in vivo tolerance tests confirmed the biocompatibility of the drug-loaded microspheres, as they did not elicit significant irritation in ocular tissues. Remarkable antibacterial effects were observed in both in vitro and in vivo experiments. Our developed PLGA microspheres show promise as an alternative therapeutic option for topical administration in managing keratitis, offering exceptional drug delivery capabilities, improved bioavailability, and potent antibacterial efficacy.

Antibacterial peptides are endogenous polypeptides produced by multicellular organisms to protect the host against pathogenic microbes, they show broad spectrum antimicrobial activities against various microorganisms and possess low propensity for developing resistance. The purpose of this study is to develop recombinant antibacterial peptide cathelicidin-BF by genetic engineering and protein engineering technology, and study its antibacterial activity in vitro and in vivo, so as to provide reference for the production and application of recombinant antibacterial peptide cathelicidin-BF. In this study, on account of Pichia pastoris eukaryotic expression system, we expressed and prepared antibacterial peptide cathelicidin-BF. Then, the minimum inhibitory concentration of antibacterial peptide cathelicidin-BF and the comparison with the antibacterial activity of antibiotics were determined through the antibacterial experiment in vitro. Chickens as infection model were used to verify the antibacterial peptide activity in vivo. The results show that the bacteriostatic ability of antibacterial peptide cathelicidin-BF is similar to that of antibiotics in certain concentration, and can reach the treatment level of antibiotics. Although the mode of administration of antibacterial peptide is still limited, this study can provide reference for the future research of antibacterial peptide.

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