Schlafens Can Put Viruses To Sleep Part 1

Abstract:

The Schlafen gene family encodes proteins involved in various biological tasks, including cell proliferation, differentiation, and T-cell development. Schlafens were initially discovered in mice, and have been studied in the context of cancer biology, as well as their role in protecting cells during viral infection. This protein family provides antiviral barriers via direct and indirect effects on virus infection. Schlafens can inhibit the replication of viruses with both RNA and DNA genomes. In this review, we summarize the cellular functions and the emerging relationship between Schlafens and innate immunity. We also discuss the functions and distinctions of this emerging family of proteins as host restriction factors against viral infection. Further research into Schlafen protein function will provide insight into the mechanisms that contribute to intrinsic and innate host immunity.

Gene family codes are closely related to immunity. Multiple gene families encode proteins and molecules representing specific immune cells that work closely together to coordinate and activate immune responses.

One of the most important gene families is the immunoglobulin family, also known as the immunoglobulin superfamily. This family encodes a series of immunoglobulin molecules, including IgG, IgM, IgA, IgE, and IgD. These immunoglobulin molecules can bind foreign antigens and activate specific immune responses. In addition, members of the immunoglobulin family can also activate and regulate the activity of immune cells by binding to receptors on the surface of immune cells.

Another important gene family is the human leukocyte antigen (HLA) family, also known as the histocompatibility complex. This family encodes human leukocyte antigens, which are present in major human tissues and can recognize and bind foreign antigens and display them to T cells in the immune system, thereby triggering a cellular immune response.

In addition, there are many other gene families encoding proteins related to immunity, such as the chemokine family, nitrite synthase family, etc.

Therefore, the proteins encoded by the gene family are critical for regulating the immune response and enhancing immunity. Mutations or variations in some genes may lead to abnormal immune function, leading to diseases such as autoimmune diseases, immunodeficiency diseases, and infectious diseases. Therefore, studying the relationship between gene family coding and immunity is of great significance for the prevention and treatment of these diseases. Therefore, we need to pay special attention to the improvement of our immunity. Cistanche can enhance immunity, and the polysaccharides in meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the bactericidal effect of immune cells.

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Keywords:

Schlafen; SLFN; innate immunity; virus; restriction factor; immune evasion.

1. Introduction

In 1998, the Schlafen (SLFN for humans; Slfn for mice) gene was first reported in the study of murine thymus development. The first Schlafens discovered were the murine genes Slfn1–4. When Slfn1 is expressed ectopically in NIH-3T3 fibroblasts, it induces G0/G1 cell cycle arrest; this observation led to the coining of the term “schlafen” from the German word meaning “to sleep” [1]. 

Later research found that Schlafens play roles in a variety of cellular functions, including anti-proliferation and cell differentiation [2–7], cancer cell migration, proliferation, and invasion prevention [8–11], sensitization of cancer cells to DNA-damaging drugs [12–17], and inhibition of viral replication [18–24]. As studies on the Schlafen family have expanded in recent years, substantial progress has been achieved toward understanding how the proteins in this family have distinct functions. Excellent recent review articles have described their significance for the field of cancer biology [25]. 

The Schlafen proteins also have roles in controlling viruses and the host immune system. Here, we address the functional similarities and differences among Schlafen family members in terms of their roles in regulating virological and immunological features. These recent findings inspire future research directions into this emerging protein family.

2. Schlafen Family Members and Protein Composition

Schlafen gene family members are highly homologous across many mammalian species. Nine Schlafen proteins are expressed in mice from chromosome 11, and six have been found in humans from chromosome 17 (Figure 1) [3,26]. Even though Slfn-like 1 (Slfn1L) is expressed on mouse chromosome 4, there is an opinion that it is not considered a ‘bona file Schlafen family member due to the extremely low similarity to Slfn genes [26,27]. In addition, Slfn6 and Slfn7 are considered to be sequences derived from either Slfn3 or Slfn4 isoforms or other mouse paralogues [1,27].

Schlafen members fall into three distinct groups, each with its unique set of characteristics and functions (Figure 1). Group I has a divergent AAA ATPase-associated domain containing a common Slfn-box region that has been termed the Schlafen core domain, and is shared with the other two groups [2,28–31]. The Schlafen core domain is horseshoe shaped and contains zinc finger motifs that are highly conserved in all members of the Schlafen family proteins. 

Groups II and III contain an additional linker domain following the Schlafen core domain, which harbors the SWADL motif defined by the amino acid sequence pattern S-W-(A/S)-(V/G/L)-D-(L/I/V) with unknown function [3,29]. Only group III proteins feature an extended carboxyl (C)-a terminal domain that matches superfamily I of DNA/RNA helicases [2]. The Schlafen core domain lacks the Walker motif. The Walker A and B represent structural motifs for nucleotide binding and were discovered in the AAA family of ATPases [32]. 

Due to the absence of Walker motifs, Schlafen proteins in groups I and II may lack ATPase activity. Putative DNA/RNA helicase domains of some group III Schlafen members have AAA domains with Walker motifs that appear to be enzymatically functional [18,33,34]. These are incomplete in murine and human Schlafen 14, which possess only the Walker B motif [31]. In addition, the C-terminal extension of some group III Schlafens possesses a nuclear localization signal (NLS) and may have nuclear functions (Figure 1) [20,24,25]

Except for the platypus, a monotreme, the Schlafen family is found in practically all mammals. Sequences similar to Schlafen genes were discovered in the amphibian Xenopus laevis and the fish species Callorhincuys milii, but not in any other non-mammalian organism. Interestingly, sequences similar to Schlafens have been found by bioinformatic analysis of genomes for orthopoxvirus (OPV), such as vaccinia, variola (smallpox), and cowpox viruses [1]. Subsequent sequencing of the camelpox virus (CMLV) identified another Schlafen-like protein called 176R. This protein consists of 502 amino acids and has a C-terminal sequence that is comparable to the Schlafen core domain of murine Schlafens. Some of these viral Schlafen (v-Slfn) genes retain an entire open reading frame (ORF). 

While the ORF for v-Slfn is intact in the genomes of camelpox, monkeypox, cowpox, mousepox, and taterapox viruses, protein expression is restricted in other OPVs, such as the vaccinia virus (VACV), due to ORF fragmentation [1,26,35]. Sequences of v-Slfn were found to be similar to the mouse and rat group I Schlafen, but lack the C-terminal domain. This implies that, while the progenitor virus of OPV may have acquired an intact Schlafen from rodents, the ORF was fragmented due to mutations acquired over time [26,27].

3. Regulated Expression of Schlafens in the Immune System

Schlafen family members have been revealed to be induced by several stimuli, including CpG-DNA [36], LPS [36–38], and pathogens, such as Brucella, Listeria [39], and rhinovirus [37]. Type I IFN and IFN receptors have been implicated in the induction of Schlafen genes, implying that Schlafens are IFN-stimulated genes (ISGs). In 2010, it was first reported that IFNα influences the expression of members of the Schlafen gene family [40]. The data presented in this study reveal that type I IFN is a potent inducer of numerous mouse Schlafen family members, including members of group I (Slfn1 and Slfn2), group II (Slfn3), and group III (Slfn5 and Slfn8). IFN-activated Stat proteins and p38 MAP kinase operated differently in their regulation of interferon-induced expression [41]. 

In Stat1 deletion in mouse embryonic fibroblasts, IFN-dependent expression of all Schlafen genes was reduced relative to parental cells, ranging from a partial reduction in Slfn3 to total transcriptional defects in Slfn1, 2, 5, and 8. Interestingly, Slfn5 expression was completely independent of Stat3, but it was increased in Stat3 knockout cells. The function of p38 MAPK-activated signaling cascades is required for the complete transcriptional activation of ISGs. However, while p38 MAPK is required for IFN-dependent expression of Schlafen genes in groups I and II, interestingly, group III gene expression is not dependent on p38 MAPK. 

In the absence of p38 MAPK, IFN-dependent mRNA expression of Slfn1, Slfn2, and to a lesser extent Slfn3, was suppressed. The group III Schlafen genes, SLFN5 and SLFN8, on the other hand, were induced by IFN in a p38 MAPK-independent manner [41]. Notably, neither Stat3 nor p38 MAPK was necessary for Slfn5 induction, indicating that alternative regulatory mechanisms are involved in this process.

The induction of ISGs by type I IFNs requires the presence of interferon-stimulated response elements (ISREs) in the promoter region of the ISG, which enables transcriptional activation via the binding of the ISGF3 transcription factor, a complex of phosphorylated STAT1/STAT2 heterodimers, and IRF9 [42]. The inducibility of Schlafens by IFNα or IFN stimuli was lower than for MxA, a conventional ISG [37]. 

Analysis of transcription factor binding sites using the MatInspector program [43] showed that MxA has six ISRE sites, whereas most human Schlafen genes have just one canonical ISRE [37]. Although the Schlafen family belongs to the group of classical ISGs regulated by the STAT complex, some Schlafens are expressed through the noncanonical IFN pathways or undefined mechanisms. Considerable levels of Schlafens are expressed in various cells, including primary fibroblasts and cancer cells, in the absence of IFN activation [18,20,44]. 

The sensitivity of Schlafen expression to IFN varies according to cell type. For example, SLFN5 expression is suppressed in malignant melanoma compared to normal melanocytes. IFNα stimulation, on the other hand, significantly increased SLFN5 expression, whereas SLFN11, SLFN12, and SLFN13 were not affected [40]. In contrast, IFN stimulants, such as poly I: C and 50 pppdsRNA, increased Slfn5 expression slightly, but not significantly, in mouse macrophage RAW 264.7 cells, whereas Slfn14 expression was significantly increased [19].

In the 50 -flanking region of the Slfn2 gene, one copy of a putative NF-κB binding site and two copies of AP-1 binding sequences are found. It has been demonstrated that CpG-DNA and LPS treatment of macrophages requires the functional interaction of NF-κB and AP1 within the promoter element [36]. Scanning the promoter region of Slfn4 with JASPAR (jaspar.cgb.ki.se) revealed the presence of AP1 and PU. 1 binding sequence, as well as two copies of IFN response elements STAT1 and IRF1 binding sequences [38]. 

In addition, a Gli1 binding site also exists within the promoter. Gli1, a Hedgehog signaling effector, is required for the activation of the Slfn4 promoter, which means that the role of Slfn4 is critical in the appearance of macrophages expressing IL1β or TNFα [45]. In cancer cells, epigenetic inhibition of gene expression via CpG promoter island hypermethylation is a frequent occurrence [46]. Several studies have reported hypermethylation of the SLFN11 gene promoter [14,46–49]. The silencing of SLFN11 by promoter CpG island hypermethylation is linked to greater resistance to platinum compounds for cancer chemotherapy [14]. Hypermethylation of a CpG promoter island inactivates SLFN11 gene expression. 

This methylation is catalyzed by two main DNA methyltransferases, DNMT1 and DNMT3B [14]. The fact that DNMT3B expression in monocytes is very low, or barely detectable [50], may imply that elevated levels of SLFN11 expression in monocytes are related to hypermethylation. It is also known from germinal center B cell differentiation studies that histone modifiers, such as EZH2 and HDACs, regulate the epigenetic expression of SLFN11 [48]. 

In addition, SLFN11 expression and the B cell lineage-specific repressor PAX5 have been shown to have a nearly perfect inverse correlation [48]. A potential PAX5 binding site (GCGTGAC) exists in the promoter region of SLFN11, suggesting that PAX5 may be one of the repressors of SLFN11 in B cells.

The Schlafen members are expressed at different phases of thymocyte development and peripheral T-cell activation in mice. Slfn1 and Slfn2 are drastically elevated during the transition from CD4 and CD8 double-positive to single-positive maturation stages. However, expression levels of both genes decrease after T cell activation [1,2,51]. Slfn3 is highly expressed in single-positive T cells throughout thymocyte development. Slfn3 is also expressed at a higher level in natural CD4+ CD25+ regulatory T cells than in CD4+ CD25− cells. Slfn3 expression is increased in CD4+ CD25- T cells upon activation but decreased in CD4+ CD25+ T cells following activation with anti-CD3/CD28 stimulation. TGF-β stimulation also decreases Slfn3 expression in the CD4+ T cell subset, suggesting that Slfn3 may be a novel marker of T cell activation [52]. Slfn4 is detected early and decreased during thymocyte development, showing the opposite phenomenon to Slfn1 [1,2]. 

Slfn4 mRNA levels are upregulated during macrophage activation, whereas they are downregulated throughout differentiation. Myelopoiesis is disrupted by constitutive Slfn4 expression in the myeloid lineage, implying that downregulation of Slfn4 gene expression during macrophage differentiation is critical, and Slfn4 may act as a modulator of this lineage [38]. Unlike the other groups, Slfn5, 8, 9, and 10 in group III do not change quantitatively during thymocyte development. During T cell activation, however, there was a significant downregulation of Slfn5 and Slfn8 expression, while Slfn9 expression increased and Slfn10 expression remained relatively constant [2]. Since SLFN14 is expressed at an exceedingly low level in T cells, it is unlikely to be linked to T cell fate [37].

The human Schlafen family is also associated with immune cell proliferation and T-cell maturation. Except for SLFN14, all human Schlafen proteins are expressed natively in monocytes, monocyte-derived dendritic cells (moDCs), and T cells [37]. The expression levels of SLFN5 in T cells, and SLFN11 in monocytes and moDCs, are notably high. The expression of SLFN5 and SLFN11 changes slightly throughout moDC differentiation. 

The expression of SLFN12L and SLFN13 is relatively modest in monocytes at rest but appears to be elevated during differentiation into moDCs, whereas SLFN12 expression is markedly reduced [37]. Thus, the downregulation and upregulation of each Schlafen family protein may represent distinct requirements for these proteins in moDC function.

Intriguingly, there appears to be a regulatory feedback mechanism for transcriptional control within the Schlafen family [53]. The loss of Slfn3 by knockout decreases Slfn4, Slfn8, and Slfn9 expression in the ileal mucosa while increasing Slfn1 and Slfn5. In addition, Slfn3 deficiency decreases Slfn4 expression and increases Slfn8 and Slfn9 expression in the thymus and spleen, where immune cells mature and/or proliferate [53]. The promoters of all members of the Schlafen family contain regions for binding of the Kruppel-like factor-6 (KLF6) transcription factor.
The NFAT-related factors ING4, ZNF333, and KLF4, are also predicted to bind to most Schlafen promoters. These transcription factors from the KLF family play different roles in gastrointestinal cell differentiation and proliferation and have different expression patterns [54]. This suggests that members of the KLF and Schlafen families may have feedback loops that act as regulators of gastrointestinal and immune cell fate in different ways [53].

4. Immunodeficiency of Schlafen Mutants

It has been observed that the Elektra mutant is a homozygous mutation of murine Slfn2 and confers vulnerability to viral and bacterial infections [55]. The mortality rate of mice after murine cytomegalovirus (MCMV) infection was significantly high compared to that of the wild-type control mice [55]. In mice with the Elektra phenotype, CD8+ and CD4+ T lymphocytes fail to expand. When compared to the wild-type cells, these cells had a higher rate of apoptosis. 

In response to T cell activation signals, this mutation is thought to cause apoptosis [9]. Elektra mice also showed a significantly lower level of T cells in response to infection with lymphocytic choriomeningitis virus. Elektra T cells, similar to recently activated T cells, fail to maintain cellular quiescence and enter a post-mitotic phase. T cells lose their proliferation potential and die in response to proliferation/activation signals, resulting in reduced T cell populations in the Elektra mutant mice [9].

There have been reports of a patient with a large heterozygous loss of the SLFN11, SLFN12, and SLFN13 genes on chromosome 17 [56]. This patient was discovered to have substantial abnormalities in T-cell proliferation and cell cycle regulation. Interestingly, the patient had upper thigh Merkel cell carcinoma, a kind of carcinoma associated with a viral infection, and was regarded to be susceptible to cancer, having been diagnosed with T cell lymphoma. 

The patient’s blood and plasma had substantial Epstein–Barr virus and Torque teno virus DNA, indicating that the patient was vulnerable to viral infections. The patient had normal CD4+/CD8+ immune cell distribution and a typical distribution of naïve and memory cells, but had aberrant T cell proliferation and excessive T cell death [56].

Mutations in SLFN14 have been linked to macrothrombocytopenia and excessive bleeding [57–61]. In addition, platelet function is diminished in patients with these mutations [61]. This SLFN14 mutation presents a species-specific phenotype, with platelet abnormalities in humans and severe microcytic erythrocytosis in mice [62]. 

Thus, SLFN14 may be an essential player in mammalian hematopoiesis and may play a role in determining platelet and erythroid lineage commitment in particular species. Furthermore, platelets are now known to have roles in a variety of innate and adaptive immunological responses, which goes far beyond the classic conception of platelets as only hemostatic and thrombolytic agents [63]. Therefore, it can be demonstrated that SLFN14 is profoundly implicated in immunological control through platelet formation and function regulation.

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