Murine Retrovirus Research Articles

Unexpected low expression of porcine endogenous retroviruses (PERVs) in porcine expanded potential stem cells (EPSCs)

Expanded potential stem cells (EPSCs) have been recently derived from porcine preimplantation embryos (Gao et al., 2019). These cells were shown to express key pluripotency genes, to be genetically stable and differentiate to derivatives of the three germ layers and additionally to trophoblast. Their molecular features and expanded potency to contribute to all embryonic and extra-embryonic cell lineages are generally not seen in the embryo-derived or induced pluripotent stem cells (iPSCs). Therefore porcine EPSCs represent a unique state of cellular potency. In the past it had been shown that human and murine embryonic stem cells (ESCs) show an increased expression of murine and human endogenous retroviruses, respectively, and retroviral expression patterns were used as markers of ESC pluripotency. An increased expression of porcine endogenous retroviruses (PERVs) was also detected in porcine iPSCs. Here we investigated 24 passages of five different clones of porcine EPSCs derived from German landrace pigs and show that they harbour PERV-A, PERV-B and PERV-C, but their expression was very low and did not change during cultivation. No recombinant PERV-A/Cs were found in these cells. The low expression despite the presence of spliced mRNA, and negative infection assay and electron microscopy results indicate that no PERV particles were released. Therefore, the absence of PERV expression seems to be a unique feature of porcine EPSCs. Most importantly, the copy number of PERV proviruses was much lower in EPSCs than in young and older pigs (29.1 copies compared with 35.8), indicating an increase in copy number during life time.

Lipid droplets are formed in 2-cell-like cells.

Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.

Identification of inflammation‑associated circulating long non‑coding RNAs and genes in intracranial aneurysm rupture‑induced subarachnoid hemorrhage.

Ruptured intracranial aneurysm (IA)-induced subarachnoid hemorrhage (SAH) triggers a series of immune responses and inflammation in the brain and body. The present study was conducted to identify additional circulating biomarkers that may serve as potential therapeutic targets for SAH-induced inflammation. Differentially expressed (DE) long non-coding RNAs (lncRNAs; DElncRNAs) and genes (DEGs) in the peripheral blood mononuclear cells between patients with IA rupture-induced SAH and healthy controls were identified in the GSE36791 dataset. DEGs were used for weighted gene co-expression network analysis (WGCNA), and SAH-associated WGCNA modules were identified. Subsequently, an lncRNA-mRNA regulatory network was constructed using the DEGs in SAH-associated WGCNA modules. A total of 25 DElncRNAs and 1,979 DEGs were screened from patients with IA-induced SAH in the GSE36791 dataset compared with the controls. A total of 11 WGCNA modules, including four upregulated modules significantly associated with IA rupture-induced SAH were obtained. The DEGs in the SAH-associated modules were associated with Gene Ontology biological processes such as ‘regulation of programmed cell death’, ‘apoptosis’ and ‘immune response’. The subsequent lncRNA-mRNA regulatory network included seven upregulated lncRNAs [HCG27, ZNFX1 antisense RNA 1, long intergenic non-protein coding RNA (LINC)00265, murine retrovirus integration site 1 homolog-antisense RNA 1, cytochrome P450 1B1-AS1, LINC01347 and LINC02193] and 375 DEGs. Functional enrichment analysis and screening in the Comparative Toxicogenomics Database demonstrated that SAH-associated DEGs, including neutrophil cytosolic factor (NCF)2 and NCF4, were enriched in ‘chemokine signaling pathway’ (hsa04062), ‘leukocyte transendothelial migration’ (hsa04670) and ‘Fc gamma R-mediated phagocytosis’ (hsa04666). The upregulated lncRNAs and genes, including NCF2 and NCF4, in patients with IA rupture-induced SAH indicated their respective potentials as anti-inflammatory therapeutic targets.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

Insights into Sensing of Murine Retroviruses.

Retroviruses are major causes of disease in animals and human. Better understanding of the initial host immune response to these viruses could provide insight into how to limit infection. Mouse retroviruses that are endemic in their hosts provide an important genetic tool to dissect the different arms of the innate immune system that recognize retroviruses as foreign. Here, we review what is known about the major branches of the innate immune system that respond to mouse retrovirus infection, Toll-like receptors and nucleic acid sensors, and discuss the importance of these responses in activating adaptive immunity and controlling infection.

P53-induced MRVI1 mediates carcinogenesis of colorectal cancer

Background: Colorectal cancer (CRC) is one of the most prevalent cancer types worldwide. Despite the advancements in current diagnosis and treatment strategies of CRC, the incidence and mortality rates of CRC have been rising. To explore novel mechanism of CRC, this study focused on the expression pattern and functional mechanism of murine retrovirus integration site 1 (MRVI1) in CRC.Methods: Tumor tissues and adjacent normal tissues were collected from CRC patients, and the expression levels of MRVI1 were determined by RT-PCR and Western blot. MRVI1 knockdown was achieved by shRNA in HCT116 and HT29 cells, followed by CCK-8 assay to detect the cell proliferation, and caspase-3 activity assay combined with nucleosome ELISA assay to detect cell apoptosis. Transwell assay was used to detect cell invasion and luciferase reporter assay was used to validate the activation of the MRVI1 promoter by p53.Results: MRVI1 was downregulated in CRC tissues and several CRC cell lines. Knockdown of MRVI1 enhanced the proliferation and apoptosis, while promoted invasion and stemness of CRC cells. Mechanism study revealed that MRVI1 was transcriptionally activated by p53 at its upstream. In addition, p53-induced inhibition of CRC prognosis depended on MRVI1.Conclusion: MRVI1 inhibited the prognosis of CRC via a mechanism involving p53 activation. MRVI1 could serve as a potential target for clinical diagnosis and treatment of CRC.

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non-syndromic form of isolated familial achalasia.

Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease. Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study. A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol: IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP3 R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entire MRVI1 gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases with MRVI1 mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform β (cGK1β) depicted a partial mislocalization of cGK1β due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1β. In individuals with an early onset of achalasia without further accompanying symptoms, MRVI1 mutations should be considered as the disease-causing defect.

Effects of Morphine on Behavioral and CNS Inflammatory Responses in GP120 Mice Under Immunocompetent vs. Immunodeficient Conditions

Abstract HIV-associated neurocognitive disorder is a common complication of HIV infection, whose development is known to be facilitated by inflammation and exacerbated by morphine. In this study, we infected HIV gp120tg mice with LP-BM5 (a murine retrovirus that can cause systemic immunodeficiency in susceptible mouse strains) to examine morphine’s effect on gp120 in both immunocompetent and immunodeficient conditions. Specifically, we examined the expression of a series of inflammation-related molecules in the brain via qRT-PCR as well as the animals’ learning/memory function using spontaneous alternation T-maze. Morphine treatment (2×25mg pellets) did not significantly affect the development of immunodeficiency induced by LP-BM5 and all brain regions examined (hippocampus, striatum, and frontal lobe) had detectable LP-BM5 viral gag genes. Morphine notably reduced the performance of gp120tg+ mice in the T-maze assay when 2-minute retention was used, regardless of LP-BM5 treatment. Without LP-BM5 treatment, morphine increased hippocampal expression of CCL2, CCL5, CXCL10, IL-12 p40, TNFα, and IFNγ, while it decreased the expression of IL-6, arginase-1, IFNα and IFNβ. For mice who were subjected to LP-BM5 treatment, hippocampal expression of CCL2, CCL5, CXCL10, IL-12p40, and IL-1β were decreased by morphine, while CCL4, TNFα, IFNγ, iNOS, arginase-1 and IFNβ were increased by morphine. Further, expression of the mu opioid receptor was upregulated by morphine without LP-BM5, yet down-regulated by morphine with LP-BM5 treatment. Altogether, the effects of morphine are complex and dependent on the immune status of the host, which will be further elucidated in the future studies. (Supported by NIH/NIDA R21DA044886 (PI, Cao))

On transposons and totipotency.

Our perception of the role of the previously considered 'selfish' or 'junk' DNA has been dramatically altered in the past 20 years or so. A large proportion of this non-coding part of mammalian genomes is repetitive in nature, classified as either satellites or transposons. While repetitive elements can be termed selfish in terms of their amplification, such events have surely been co-opted by the host, suggesting by itself a likely altruistic function for the organism at the subject of such natural selection. Indeed numerous examples of transposons regulating the functional output of the host genome have been documented. Transposons provide a powerful framework for large-scale relatively rapid concerted regulatory activities with the ability to drive evolution. Mammalian totipotency has emerged as one key stage of development in which transposon-mediated regulation of gene expression has taken centre stage in the past few years. During this period, large-scale (epigenetic) reprogramming must be accomplished in order to activate the host genome. In mice and men, one particular element murine endogenous retrovirus with leucine tRNA primer (MERVL) (and its counterpart human ERVL (HERVL)) appears to have acquired roles as a key driving force in this process. Here, I will discuss and interpret the current knowledge and its implications regarding the role of transposons, particularly of long interspersed nuclear elements (LINE-1s) and endogenous retroviruses (ERVs), in the regulation of totipotency. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA

ABSTRACTCellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukaemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending on the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

The Mechanisms Linking Relative Hypercortisolism - The Common Feature Across COVID-19 Risks - To ARDS, Septic Shock, and Cytokine Dysregulation

Covid-19 mortality risk factors share a common feature: a high ratio of cortisol to DHEA, referred to as relative hypercortisolism (RHC).Starting from a low just after age 25, adult COVID-19 mortality rates rise in tandem with this ratio as DHEA levels fall by over 80% by age 70. Conversely, juvenile COVID-19 morbidity rates _fall_ with this ratio as DHEA rises to pre-adolescent levels after age 6. Moreover, RHC predicts the severity and likelihood of death for both community acquired pneumonia and septic shock, the primary COVID-19 modes of death. Consistent with this finding, DHEA prevents cytokinedysregulation in a murine leukemia retrovirus model. DHEA, byinhibiting the PI3K/AKT pathway, also inhibits viral replication.DHEA, antagonized by cortisol, maintains intracellular calciumhomeostasis, a critical function during viral infections. The cortisol to DHEA ratio determines the exon splicing of the calcium-sensitive big potassium (BKCa) channel, which exerts predominate control over cell and inner mitochondrial membrane polarization. DHEA increases BKCa calcium sensitivity, and thereby reduces calcium influx into the cell and mitochondrial matrix. This effect–together with vitamin D3/calcitriol promoted calbindin-D9k/28k, magnesium, andubiquinone–protects against mitochondrial calcium overload. Absent these key factors, the COVID-19 virus, SARS-CoV-2, like other viruses disrupts intracellular calcium homeostasis to trigger mitochondrial calcium overload in susceptible hosts. The virus then hijacks the inflammatory ER stress and unfolded protein response (UPR) for its own replication.UPR underlies the virus’s lethality. It splices X-box protein 1(XBP-1) to its active form, which in turn activates the transcription regulator SNAI1. sXBP-1 and SNAI1 shift cellular metabolism from the pentose phosphate pathway to the hexosamine biosynthesis pathway and induce insulin resistance. SNAI1 also causes epidermal-to-mesenchymal transition (EMT). In this process, SNAI1 represses transcription of epithelial and vascular endothelial cadherins along with othercritical proteins forming the tight junction barriers in the lung and vasculature. UPR also inhibits expression of the epithelial sodium channel (ENaC), which maintains alveolar fluid balance. DHEAdose-dependently reduces XBP-1 mRNA splicing in cultured hepatocytes treated with the ER stress/UPR inducer tunicamycin.RHC and insulin resistance also produce a pathological immuneresponse. Insulin resistance via mTORC1 inhibits the cell energy sensor AMPK and, consequently, the first line of defense against the virus, autophagy. Severe mitochondrial calcium overload associated with RHC oxidizes phospholipids and predisposes the cell to necrosis.Upon cell lysis, the oxidized phospholipids trigger sterileinflammation, drawing cytokine-secreting neutrophils and macrophages to the site. Further exacerbating inflammation, insulin resistance and its inhibition of PPAR-γ skews macrophage polarization to thepro-inflammatory M1 phenotype. Meanwhile, RHC promotes glucocorticoid recycling via 11β-HSD1 in thymocytes. This recycling causesactivation-induced cell death, suppresses lymphocyte countproliferation, and leads to IL-1β hypersecretion and worsening RHC.Ultimately, this cytokine storm and severe RHC induce autoimmunity.Evidence suggests this pathological state can be avoided by restoring calcium homeostasis using a combination of DHEA, vitaminD3/calcitriol, magnesium, ubiquinone, and the AMPK/SIRT1 activators, acetyl L-carnitine and alpha lipoic acid.

The MRVI1-AS1/ATF3 signaling loop sensitizes nasopharyngeal cancer cells to paclitaxel by regulating the Hippo-TAZ pathway.

Long non-coding RNA (lncRNA) plays an important role in malignant tumor occurrence, development, and chemoresistance, but the mechanism of how they affect nasopharyngeal cancer (NPC) paclitaxel chemosensitivity is unclear. In this study, lncRNA array of CNE-1 and HNE-2 paclitaxel-resistant cells and their parental strains revealed that the paclitaxel-resistant strains had significantly lower MRVI1-AS1 (murine retrovirus integration site 1 homolog antisense RNA 1) expression than the parental strains, and that MRVI1-AS1 overexpression in vitro and in vivo increased paclitaxel chemosensitivity. Further, MRVI1-AS1 upregulated ATF3 (activating transcription factor 3) by simultaneously inhibiting miR-513a-5p (microRNA-513a-5p) and miR-27b-3p expression levels to increase NPC paclitaxel chemosensitivity. Chromatin immunoprecipitation and quantitative real-time PCR showed that ATF3 could feed-back MRVI1-AS1 regulation positively. Furthermore, MRVI1-AS1 and ATF3 could form a positive feedback loop, which promoted the expression of RASSF1 (Ras association domain family member 1), a Hippo-TAZ (tafazzin) signaling pathway regulatory factor, thereby inhibiting TAZ expression. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and flow cytometry showed that the decreased TAZ increased NPC cell paclitaxel chemosensitivity. Overall, the results indicate that the MRVI1-AS1/ATF3 signaling pathway can increase NPC paclitaxel chemosensitivity by modulating the Hippo-TAZ signaling pathway. Therefore, targeting the loop may be a new NPC treatment strategy.

Antiallodynic Effects of Cannabinoid Receptor 2 (CB2R) Agonists on Retrovirus Infection-Induced Neuropathic Pain.

The most common neurological complication in patients receiving successful combination antiretroviral therapy (cART) is peripheral neuropathic pain. Data show that distal symmetric polyneuropathy (DSP) also develops along with murine acquired immunodeficiency syndrome (MAIDS) after infection with the LP-BM5 murine retrovirus mixture. Links between cannabinoid receptor 2 (CB2R) and peripheral neuropathy have been established in animal models using nerve transection, chemotherapy-induced pain, and various other stimuli. Diverse types of neuropathic pain respond differently to standard drug intervention, and little is currently known regarding the effects of modulation through CB2Rs. In this study, we evaluated whether treatment with the exogenous synthetic CB2R agonists JWH015, JWH133, Gp1a, and HU308 controls neuropathic pain and neuroinflammation in animals with chronic retroviral infection. Hind-paw mechanical hypersensitivity in CB2R agonist-treated versus untreated animals was assessed using the MouseMet electronic von Frey system. Multicolor flow cytometry was used to determine the effects of CB2R agonists on macrophage activation and T-lymphocyte infiltration into dorsal root ganglia (DRG) and lumbar spinal cord (LSC). Results demonstrated that, following weekly intraperitoneal injections starting at 5 wk p.i., JWH015, JWH133, and Gp1a, but not HU308 (5 mg/kg), significantly ameliorated allodynia when assessed 2 h after ligand injection. However, these same agonists (2x/wk) did not display antiallodynic effects when mechanical sensitivity was assessed 24 h after ligand injection. Infection-induced macrophage activation and T-cell infiltration into the DRG and LSC were observed at 12 wk p.i., but this neuroinflammation was not affected by treatment with any CB2R agonist. Activation of JAK/STAT3 has been shown to contribute to development of neuropathic pain in the LSC and pretreatment of primary murine microglia (2 h) with JWH015-, JWH133-, or Gp1a-blocked IFN-gamma-induced phosphorylation of STAT1 and STAT3. Taken together, these data show that CB2R agonists demonstrate acute, but not long-term, antiallodynic effects on retrovirus infection-induced neuropathic pain.

Peptides of endogenous retrovirus Gag gene variants are not agonistic ligands for autoreactive T cells in non-obese diabetic mice

Abstract Endogenous retroviruses (ERV) have been isolated from the pancreatic islets of non-obese diabetic (NOD) mice. We previously demonstrated that a murine leukemia retrovirus–like ERV Gag protein was secreted together with exosomes by an insulinoma and by primary islet mesenchymal stem cells. Here, we further demonstrated that Gag is an effective antigen to stimulate IFN-gamma production by autoreactive T cells using recombinant Gag protein or virus-like particles (VLPs) expressing the Gag gene. However, candidate peptides of this Gag protein were not effective in stimulating the T cells. Upon analyses of ERV Gag genes expressed in the islets of prediabetic NOD mice, we were surprised to find multiple Gag gene variants with complete open-reading frames. These results suggested the expression of different endogenous proviruses, or alternatively, the detection of point mutations incurred during viral replication. A new batch of altered peptides was synthesized reflecting the variations among the Gag gene variants, and a few of them induced detectable amount of cytokine secretion from the T cells. But, the levels of IFN-gamma induced by the peptides were much lower than that induced by the Gag protein and the VLPs, suggesting a low affinity activation. This weak T-cell-stimulating activity of the Gag peptides was further confirmed by testing diabetogenic T-cell clones, as well as by using MHC tetramers loaded with candidate peptides. The data suggest that ERV Gag or its gene variants may trigger autoimmunity via a pathway that could be independent from direct presentation of viral peptides to autoreactive T cells.

HIF-1α is essential for T cell suppression by murine LP-BM5 retrovirus infection-augmented Monocytic Myeloid Derived Suppressor Cells

Abstract Compared to naïve mice, monocytic myeloid-derived suppressor cells (M-MDSCs) increase in number, and suppressive efficiency, after infection with the immunodeficiency causing mouse retrovirus, LP-BM5. In ex vivo suppression assays, M-MDSCs, enriched from spleens of LP-BM5 infected mice inhibited both T- and as a novel finding, B-cell responsiveness. For M-MDSC suppression of T-cell proliferation and IFN-gamma production, we previously reported the mechanism to be almost completely iNOS/NO dependent. In contrast, the suppression of B-cell proliferation and IL-10 secretion by predominantly T2-B cells was only ~50% iNOS/NO dependent, with V-domain Ig suppressor of T cell activation (VISTA), superoxide and peroxynitrite also playing substantial effector roles. In light of recent reports on the Hypoxia-inducible transcription factor 1, α subunit (HIF-1α) regulating the function and differentiation of MDSCs, and reports of its role as a principal regulator of NO production, we describe here the characterization of M-MDSCs from myeloid-specific HIF-1α deficient, vs. w.t., B6 mice. As early as 5 weeks post infection, enriched M-MDSC preparations from HIF-1α deficient mice were significantly impaired functionally, in some cases with a total inability, to suppress in vitro T cell responsiveness. However, these same M-MDSC preparations suppressed B-cell responsiveness at a level very similar to that of control w.t. M-MDSCs. Experiments are in progress to further understand the role HIF-1α plays in the impairment of M-MDSC ex vivo suppression of T cell proliferation and function, including a possibility that HIF-1α regulates iNOS/NO production in M-MDSCs only when in the process of suppressing T- but not B-cell targets.

Infection of B Cell Follicle-Resident Cells by Friend Retrovirus Occurs during Acute Infection and Is Maintained during Viral Persistence.

B cell follicles of the spleen and lymph nodes are immune privileged sites and serve as sanctuaries for infected CD4+ cells in HIV infection. It is assumed that CD8+ T cell responses promote the establishment of the reservoir, as B cell follicles do not permit CD8+ T cell entry. Here we analyzed the infected cell population in the Friend retrovirus (FV) infection and investigated whether FV can similarly infect follicular cells. For analysis of FV-infected cells, we constructed a recombinant FV encoding the bright fluorescent protein mWasabi and performed flow cytometry with cells isolated from spleens, lymph nodes and bone marrow of FV-mWasabi-infected mice. Using t-stochastic neighbor embedding for data exploration, we demonstrate how the target cell population changes during the course of infection. While FV was widely distributed in erythrocytes, myeloid cells, B cells, and CD4+ T cells in the acute phase of infection, the bulk viral load in the late phase was carried by macrophages and follicular B and CD4+ T cells, suggesting that FV persists in cells that are protected from CD8+ T cell killing. Importantly, seeding into follicular cells was equally observed in CD8+ T cell-depleted mice and in highly FV-susceptible mice that mount a very weak immune response, demonstrating that infection of follicular cells is not driven by immune pressure. Our data demonstrate that infection of cells in the B cell follicle is a characteristic of the FV infection, making this murine retrovirus an even more valuable model for development of retrovirus immunotherapy approaches.IMPORTANCE Human immunodeficiency virus is notorious for its ability to avoid clearance by therapeutic interventions, which is partly attributed to the establishment of reservoirs in latently infected cells and cells that reside in immunologically privileged B cell follicles. In the work presented here, we show that cells of the B cell follicle are equally infected by a simple mouse gammaretrovirus. Using fluorescently labeled Friend retrovirus, we found that B cells and T cells in the B cell follicle, while not carrying the bulk of the virus load, were indeed infected by Friend virus in the early acute phase of the infection and persisted in the chronic infection. Our results suggest that infection of follicular cells may be a shared property of lymphotropic viruses and propose the FV infection of mice as a useful model to study strategies for follicular reservoir elimination.

Circulating Y-RNAs in Extracellular Vesicles and Ribonucleoprotein Complexes; Implications for the Immune System.

The exchange of extracellular vesicles (EV) between immune cells plays a role in various immune regulatory processes. EV are nano-sized lipid bilayer-enclosed structures that contain a multitude of proteins and small non-coding RNA molecules. Of the various RNA classes present in EV, miRNAs have been most intensively studied because of their known gene-regulatory functions. These miRNAs constitute only a minor part of all EV-enclosed RNA, whereas other 20–200 nt sized non-coding RNAs were shown to be abundantly present in EV. Several of these mid-sized RNAs perform basic functions in cells, but their function in EV remains elusive. One prominent class of mid-sized extracellular RNAs associated with EV are the Y-RNAs. This family of highly conserved non-coding RNAs was initially discovered as RNA component of circulating ribonucleoprotein autoantigens in serum from Systemic Lupus Erythematosus and Sjögren's Syndrome patients. Y-RNA has been implicated in cellular processes such as DNA replication and RNA quality control. In recent years, Y-RNA has been abundantly detected in EV from multiple different cell lines and biofluids, and also in murine and human retroviruses. Accumulating evidence suggests that EV-associated Y-RNA may be involved in a range of immune-related processes, including inflammation, immune suppression, and establishment of the tumor microenvironment. Moreover, changes in plasma levels of extracellular Y-RNA have been associated with various diseases. Recent studies have aimed to address the mechanisms underlying their release and function. We for example showed that the levels of EV-associated Y-RNA released by immune cells can be regulated by Toll-like receptor (TLR) signaling. Combined, these data have triggered increased interest in extracellular Y-RNAs. In this review, we provide an overview of studies reporting the occurrence of extracellular Y-RNAs, as well as signaling properties and immune-related functions attributed to these RNAs. We list RNA-binding proteins currently known to interact with Y-RNAs and evaluate their occurrence in EV. In parallel, we discuss technical challenges in assessing whether extracellular Y-RNAs are contained in ribonucleoprotein complexes or EV. By integrating the current knowledge on extracellular Y-RNA we further reflect on the biomarker potential of Y-RNA and their role in immune cell communication and immunopathology.

A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus

SummaryLymph- and blood-borne retroviruses exploit CD169/Siglec-1-mediated capture by subcapsular sinus and marginal zone metallophilic macrophages for trans-infection of permissive lymphocytes. However, the impact of CD169-mediated virus capture on retrovirus dissemination and pathogenesis in vivo is unknown. In a murine model of the splenomegaly-inducing retrovirus Friend virus complex (FVC) infection, we find that while CD169 promoted draining lymph node infection, it limited systemic spread to the spleen. At the spleen, CD169-expressing macrophages captured incoming blood-borne retroviruses and limited their spread to the erythroblasts in the red pulp where FVC manifests its pathogenesis. CD169-mediated retroviral capture activated conventional dendritic cells 1 (cDC1s) and promoted cytotoxic CD8+ T cell responses, resulting in efficient clearing of FVC-infected cells. Accordingly, CD169 blockade led to higher viral loads and accelerated death in susceptible mouse strains. Thus, CD169 plays a protective role during FVC pathogenesis by reducing viral dissemination to erythroblasts and eliciting an effective cytotoxic T lymphocyte response via cDC1s.

Ancient Evolutionary Origin and Positive Selection of the Retroviral Restriction Factor Fv1 in Muroid Rodents.

The laboratory mouse Fv1 gene encodes a retroviral restriction factor that mediates resistance to murine leukemia viruses (MLVs). Sequence similarity between Fv1 and the gag protein of the murine endogenous retrovirus L (MuERV-L) family of ERVs suggests that Fv1 was coopted from an ancient provirus. Previous evolutionary studies found Fv1 orthologs only in the genus Mus Here, we describe identification of orthologous Fv1 sequences in several species belonging to multiple families of rodents outside the genus Mus We show that these Fv1 orthologs are in the same region of conserved synteny, between the genes Miip and Mfn2, suggesting a minimum insertion time of 45 million years for the ancient progenitor of Fv1 Our analysis also revealed that Fv1 was not detectable or heavily mutated in some lineages in the superfamily Muroidea, while, in concert with previous findings in the genus Mus, we found strong evidence of positive selection of Fv1 in the African clade in the subfamily Muridae Residues identified as evolving under positive selection include those that have been previously found to be important for restriction of multiple retroviral lineages. Taken together, these findings suggest that the evolutionary origin of Fv1 substantially predates Mus evolution, that the rodent Fv1 has been shaped by lineage-specific differential selection pressures, and that Fv1 has long been evolving under positive selection in the rodent family Muridae, supporting a defensive role that significantly antedates exposure to MLVs.IMPORTANCE Retroviruses have adapted to living in concert with their hosts throughout vertebrate evolution. Over the years, the study of these relationships revealed the presence of host proteins called restriction factors that inhibit retroviral replication in host cells. The first of these restriction factors to be identified, encoded by the Fv1 gene found in mice, was thought to have originated in the genus Mus In this study, we utilized genome database searches and DNA sequencing to identify Fv1 copies in multiple rodent lineages. Our findings suggest a minimum time of insertion into the genome of rodents of 45 million years for the ancestral progenitor of Fv1 While Fv1 is not detectable in some lineages, we also identified full-length orthologs showing signatures of a molecular "arms race" in a family of rodent species indigenous to Africa. This finding suggests that Fv1 in these species has been coevolving with unidentified retroviruses for millions of years.

Acoustic waves improves retroviral transduction in human retinal stem cells

BackgroundsThe plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. MethodsRSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 μg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. ResultsOur results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 μg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 μg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. ConclusionWe demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.