MX2 forms nucleoporin-comprising cytoplasmic biomolecular condensates that lure viral capsids.

Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1.

First-principles prediction of high carrier mobility for ß-phase MX2N4 (M = Mo, W; X = Si, Ge) monolayers.

The recently synthesized monolayer MoSi2N4 (Science 2020, 369, 367) exhibits exceptional environmental stability, a moderate band gap, and excellent mechanical properties, presenting exciting opportunities for the exploration of two-dimensional (2D) MX2Z4 materials. However, the low carrier mobility of α-phase MoSi2N4 significantly limits its potential applications in field-effect transistor (FET) devices. In this study, we systematically investigate the structural stability, elastic properties, and carrier mobility of a novel family of ß-phase MX2N4 (M = Mo, W; X = Si, Ge) monolayers through first-principles calculations. Our findings reveal that these ß-phase MX2N4 monolayers demonstrate remarkable dynamic, thermal, and mechanical stability. Specifically, we identify the MoSi2N4, MoGe2N4, WSi2N4, and WGe2N4 monolayers as semiconductors with band gaps of 2.70 eV, 1.57 eV, 3.12 eV, and 1.93 eV, respectively, as calculated using the HSE06 functional. Moreover, the MX2N4 monolayers exhibit significant elastic anisotropy, characterized by high ideal tensile strengths and a critical tensile strain exceeding 25%. Notably, the WGe2N4 monolayer displays exceptional anisotropic in-plane charge transport, achieving mobility levels of up to 104 cm2V- 1S- 1, surpassing those of the α-phase MX2N4 monolayers. These novel ternary monolayer structures have the potential to broaden the 2D MX2Z4 material family and emerge as promising candidates for applications in field-effect transistors.

The avengers: SAMHD1 cooperates with MX2/MxB to defend against HIV-1.

SAMHD1 is an intrinsic limiting factor that effectively prevents HIV-1 infection in macrophages, dendritic cells, and resting CD4+ T cells. Extensive studies have underscored the indispensable role of the dNTPase activity of SAMHD1 in its antiviral function by primarily depleting dNTPs in quiescent cells, thereby impeding HIV-1 cDNA synthesis. However, recent advancements in understanding posttranslational modifications of SAMHD1 have revealed specific modification site mutants that maintain their ability to reduce dNTP levels while impairing the inhibition of HIV-1 replication. Thus, the precise anti-HIV-1 mechanism of SAMHD1 remains enigmatic, necessitating a comprehensive understanding of the underlying mechanisms to develop novel therapeutic strategies targeting its antiviral activity. Recent findings by Guo et al. shed light on the role of SAMHD1 as an HIV-1 core sensor in suppressing HIV-1 infection after viral cDNA synthesis through its interaction with MX2 (H. Guo, W. Yang, H. Li, J. Yang, et al., mBio 15:e01363-24, 2024, https://doi.org/10.1128/mbio.01363-24).

Evaluation of the effect of sustained-release progesterone injection on the expression of interferon-related genes in repeat-breeder dairy cows.

BACKGROUND: Repeat-breeder cows repeatedly fail to conceive after at least three attempts and return to oestrus at apparently normal intervals. Repeat-breeder cows cause economic losses in dairy farms in different ways. OBJECTIVE: In the present study, we investigated the effect of sustained-release progesterone injection in two different doses on the expression of interferon-related genes in repeat-breeder dairy cows. METHODS: A total of 96 repeat-breeder primiparous and multiparous cows were assigned among three groups: control group, inseminated and do not receive progesterone treatment; P400 and P600 groups, inseminated and received a single-intramuscular injection of 400 and 600 mg slow-release progesterone 5 days after insemination, respectively. Blood sampling was carried out on Day 20 after AI for progesterone measurement and evaluation of gene expression for ISG15, MX1 and MX2 genes. RESULTS: One injection of sustained-release progesterone increased the expression of ISG15, MX1 and MX2 genes with differences between two different progesterone concentrations. For all three genes, the level of gene expression was higher in progesterone-supplemented group than in control group, when P400 and P600 groups considered together. The level of MX2 gene expression was significantly higher in pregnant cows than non-pregnant cows. There was a significant positive correlation between expression level of all three genes and blood progesterone concentration. The expression level of ISG15 gene showed a significant positive correlation with MX1 and MX2 gene expression. CONCLUSION: The use of this sustained-release progesterone is simple and can be used in repeat-breeder cows to improve fertility.

The SAMHD1-MX2 axis restricts HIV-1 infection at postviral DNA synthesis.

HIV-1 replication is tightly regulated in host cells, and various restriction factors have important roles in inhibiting viral replication. SAMHD1, a well-known restriction factor, suppresses HIV-1 replication by hydrolyzing intracellular dNTPs, thereby limiting the synthesis of viral cDNA in quiescent cells. In this study, we revealed an additional and distinct mechanism of SAMHD1 inhibition during the postviral cDNA synthesis stage. Using immunoprecipitation and mass spectrometry analysis, we demonstrated the interaction between SAMHD1 and MX2/MxB, an interferon-induced antiviral factor that inhibits HIV-1 cDNA nuclear import. The disruption of endogenous MX2 expression significantly weakened the ability of SAMHD1 to inhibit HIV-1. The crucial region within SAMHD1 that binds to MX2 has been identified. Notably, we found that SAMHD1 can act as a sensor that recognizes and binds to the incoming HIV-1 core, subsequently delivering it to the molecular trap formed by MX2, thereby blocking the nuclear entry of the HIV-1 core structure. SAMHD1 mutants unable to recognize the HIV-1 core showed a substantial decrease in antiviral activity. Certain mutations in HIV-1 capsids confer resistance to MX2 inhibition while maintaining susceptibility to suppression by the SAMHD1-MX2 axis. Overall, our study identifies an intriguing antiviral pattern wherein two distinct restriction factors, SAMHD1 and MX2, collaborate to establish an alternative mechanism deviating from their actions. These findings provide valuable insight into the complex immune defense networks against exogenous viral infections and have implications for the development of targeted anti-HIV therapeutics. IMPORTANCE: In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense.

Knockdown of ADAM8 inhibits the proliferation, migration, invasion, and tumorigenesis of renal clear cell carcinoma cells to enhance the immunotherapy efficacy.

The current study performed bioinformatics and in vitro and in vivo experiments to explore the effects of ADAM8 on the malignant behaviors and immunotherapeutic efficacy of renal clear cell carcinoma (ccRCC) Cells. The modular genes most associated with immune cells were screened. Then, prognostic risk models were constructed by univariate COX analysis, LASSO regression analysis and multivariate COX analysis, and their diagnostic value was determined. The correlation between tumor mutation load (TMB) scores and the prognosis of ccRCC patients was clarified. Finally, six key genes (ABI3, ADAM8, APOL3, MX2, CCDC69, and STAC3) were analyzed for immunotherapy efficacy. Human and mouse ccRCC cell lines and human proximal tubular epithelial cell lines were used for in vitro cell experiments. The effect of ADAM8 overexpression or knockdown on tumor formation and survival in ccRCC cells was examined by constructing subcutaneous transplanted tumor model. Totally, 636 Black module genes were screened as being most associated with immune cell infiltration. Six genes were subsequently confirmed for the construction of prognostic risk models, of which ABI3, APOL3 and CCDC69 were low-risk factors, while ADAM8, MX2 and STAC3 were high-risk factors. The constructed risk model based on the identified six genes could accurately predict the prognosis of ccRCC patients. Besides, TMB was significantly associated with the prognosis of ccRCC patients. Furthermore, ABI3, ADAM8, APOL3, MX2, CCDC69 and STAC3 might play important roles in treatment concerning CTLA4 inhibitors or PD-1 inhibitors or combined inhibitors. Finally, we confirmed that ADAM8 could promote the proliferation, migration and invasion of ccRCC cells through in vitro experiments, and further found that in in vivo experiments, ADAM8 knockdown could inhibit tumor formation in ccRCC cells, improve the therapeutic effect of anti-PD1, and prolong the survival of mice. Our study highlighted the alleviative role of silencing ADAM8 in ccRCC patients.

Circ_ATAD3B inhibits cell proliferation of breast cancer via mediating the miR-570-3p/MX2 axis.

It has been discovered that some circular RNAs can serve as excellent therapeutic targets for breast cancer (BC). However, the biological role that circ ATAD3B plays in BC is not yet completely understood. As a result, the purpose of this work was to evaluate the function of circ_ATAD3B in the development of BC. Three different GEO datasets were used to compile the expression profiles of circRNAs related to BC (GSE101124, GSE165884, and GSE182471). CCK-8 and the production of clones, in addition to RT-PCR and western blot assays, were utilized in this study to evaluate the regulation of these three biological molecules in the process of BC carcinogenesis.circ_ATAD3B was the only potential BC-related circRNA that was significantly reduced in BC tumor tissues, and it functioned as a miR-570-3p sponge to suppress cell survival and proliferation, as stated by the aforementioned two algorithms. The expression of MX2 was boosted when circ_ATAD3B was used to sponge miR-570-3p. The inhibitory effect that circ_ATAD3B has on the malignant phenotype of BC cells was overcome by the expression of miR-570-3p through up-regulation and MX2 through down-regulation. The tumor suppressor circ_ATAD3B prevents cancer progression by regulating the miR-570-3p/MX2 pathway. Circ_ATAD3B may be a candidate for targeted therapy of breast cancer.

You Shall Not Pass: MX2 Proteins Are Versatile Viral Inhibitors.

Myxovirus resistance (MX) proteins are pivotal players in the innate immune response to viral infections. Less than 10 years ago, three independent groups simultaneously showed that human MX2 is an interferon (IFN)-stimulated gene (ISG) with potent anti-human immunodeficiency virus 1 (HIV-1) activity. Thenceforth, multiple research works have been published highlighting the ability of MX2 to inhibit RNA and DNA viruses. These growing bodies of evidence have identified some of the key determinants regulating its antiviral activity. Therefore, the importance of the protein amino-terminal domain, the oligomerization state, or the ability to interact with viral components is now well recognized. Nonetheless, there are still several unknown aspects of MX2 antiviral activity asking for further research, such as the role of cellular localization or the effect of post-translational modifications. This work aims to provide a comprehensive review of our current knowledge on the molecular determinants governing the antiviral activity of this versatile ISG, using human MX2 and HIV-1 inhibition as a reference, but drawing parallelisms and noting divergent mechanisms with other proteins and viruses when necessary.

The m6A-regulation and single cell effect pattern in sunitinib resistance on clear cell renal cell carcinoma: Identification and validation of targets.

Background: Sunitinib is the main target drug for clear cell renal cell carcinoma. However, the effect of sunitinib is often limited by acquired drug resistance. Methods: The open-accessed data used in this study were obtained from different online public databases, which were analyzed using the R software. The RNA level of specific genes was detected using quantitative Real-Time PCR. Sunitinib-resistant cell lines were constructed based on protocol get from the previous study. Colony formation and Cell Counting Kit-8 assays were applied to detect cell proliferation ability. Results: In this study, through publicly available data and high-quality analysis, we deeply explored the potential biological mechanisms that affect the resistance of sunitinib. Detailed, data from GSE64052, GSE76068 and The Cancer Genome Atlas were extracted. We identified the IFITM1, IL6, MX2, PCOLCE2, RSAD2 and SLC2A3 were associated with sunitinib resistance. Single-cell analysis, prognosis analysis and m6A regulatory network were conducted to investigate their role. Moreover, the MX2 was selected for further analysis, including its biological role and effect on the ccRCC microenvironment. Interestingly, we noticed that MX2 might be an immune-related gene that could affect the response rate of immunotherapy. Then, in vitro experiments validated the overexpression of MX2 in sunitinib-resistance cells. Colony formation assay indicated that the knockdown of MX2 could remarkably inhibit the proliferation ability of 786-O-Res and Caki-1-Res when exposed to sunitinib. Conclusion: In summary, through publicly available data and high-quality analysis, we deeply explored the potential biological mechanisms that affect the resistance of sunitinib. MX2 was selected for further analysis, including its biological role and effect on the ccRCC microenvironment. Finally, in vitro experiments were used to validate its role in ccRCC.

Stress-Tuned Optical Transitions in Layered 1T-MX2 (M=Hf, Zr, Sn; X=S, Se) Crystals.

Optical measurements under externally applied stresses allow us to study the materials' electronic structure by comparing the pressure evolution of optical peaks obtained from experiments and theoretical calculations. We examine the stress-induced changes in electronic structure for the thermodynamically stable 1T polytype of selected MX2 compounds (M=Hf, Zr, Sn; X=S, Se), using the density functional theory. We demonstrate that considered 1T-MX2 materials are semiconducting with indirect character of the band gap, irrespective to the employed pressure as predicted using modified Becke-Johnson potential. We determine energies of direct interband transitions between bands extrema and in band-nesting regions close to Fermi level. Generally, the studied transitions are optically active, exhibiting in-plane polarization of light. Finally, we quantify their energy trends under external hydrostatic, uniaxial, and biaxial stresses by determining the linear pressure coefficients. Generally, negative pressure coefficients are obtained implying the narrowing of the band gap. The semiconducting-to-metal transition are predicted under hydrostatic pressure. We discuss these trends in terms of orbital composition of involved electronic bands. In addition, we demonstrate that the measured pressure coefficients of HfS2 and HfSe2 absorption edges are in perfect agreement with our predictions. Comprehensive and easy-to-interpret tables containing the optical features are provided to form the basis for assignation of optical peaks in future measurements.

MX2: Identification and systematic mechanistic analysis of a novel immune-related biomarker for systemic lupus erythematosus.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs. However, the current SLE-related biomarkers still lack sufficient sensitivity, specificity and predictive power for clinical application. Thus, it is significant to explore new immune-related biomarkers for SLE diagnosis and development. Methods: We obtained seven SLE gene expression profile microarrays (GSE121239/11907/81622/65391/100163/45291/49454) from the GEO database. First, differentially expressed genes (DEGs) were screened using GEO2R, and SLE biomarkers were screened by performing WGCNA, Random Forest, SVM-REF, correlation with SLEDAI and differential gene analysis. Receiver operating characteristic curves (ROCs) and AUC values were used to determine the clinical value. The expression level of the biomarker was verified by RT‒qPCR. Subsequently, functional enrichment analysis was utilized to identify biomarker-associated pathways. ssGSEA, CIBERSORT, xCell and ImmuCellAI algorithms were applied to calculate the sample immune cell infiltration abundance. Single-cell data were analyzed for gene expression specificity in immune cells. Finally, the transcriptional regulatory network of the biomarker was constructed, and the corresponding therapeutic drugs were predicted. Results: Multiple algorithms were screened together for a unique marker gene, MX2, and expression analysis of multiple datasets revealed that MX2 was highly expressed in SLE compared to the normal group (all P < 0.05), with the same trend validated by RT‒qPCR (P = 0.026). Functional enrichment analysis identified the main pathway of MX2 promotion in SLE as the NOD-like receptor signaling pathway (NES=2.492, P < 0.001, etc.). Immuno-infiltration analysis showed that MX2 was closely associated with neutrophils, and single-cell and transcriptomic data revealed that MX2 was specifically expressed in neutrophils. The NOD-like receptor signaling pathway was also remarkably correlated with neutrophils (r >0.3, P < 0.001, etc.). Most of the MX2-related interacting proteins were associated with SLE, and potential transcription factors of MX2 and its related genes were also significantly associated with the immune response. Conclusion: Our study found that MX2 can serve as an immune-related biomarker for predicting the diagnosis and disease activity of SLE. It activates the NOD-like receptor signaling pathway and promotes neutrophil infiltration to aggravate SLE.

Challenges of Wafer-Scale Integration of 2D Semiconductors for High-Performance Transistor Circuits.

Large-area 2D-material-based devices may find applications as sensor or photonics devices or can be incorporated in the back end of line (BEOL) to provide additional functionality. The introduction of highly scaled 2D-based circuits for high-performance logic applications in production is projected to be implemented after the Si-sheet-based CFET devices. Here, a view on the requirements needed for full wafer integration of aggressively scaled 2D-based logic circuits, the status of developments, and the definition of the gaps to be bridged is provided. Today, typical test vehicles for 2D devices are single-sheet devices fully integrated in a lab environment, but transfer to a more scaled device in a fab environment has been demonstrated. This work reviews the status of the module development, including considerations for setting up fab-compatible process routes for single-sheet devices. While further development on key modules is still required, substantial progress is made for MX2 channel growth, high-k dielectric deposition, and contact engineering. Finally, the process requirements for building ultra-scaled stacked nanosheets are also reflected on.

MX2 Viral Substrate Breadth and Inhibitory Activity Are Regulated by Protein Phosphorylation.

Human immunodeficiency virus type-1 (HIV-1) infection is potently inhibited by human myxovirus resistance 2 (MX2/MxB), which binds to the viral capsid and blocks the nuclear import of viral DNA. We have recently shown that phosphorylation is a key regulator of MX2 antiviral activity, with phosphorylation of serine residues at positions 14, 17, and 18 repressing MX2 function. Here, we extend the study of MX2 posttranslational modifications and identify serine and threonine phosphorylation in all domains of MX2. By substituting these residues with aspartic acid or alanine, hence mimicking the presence or absence of a phosphate group, respectively, we identified key positions that control MX2 antiviral activity. Aspartic acid substitutions of residues Ser306 or Thr334 and alanine substitutions of Thr343 yielded proteins with substantially reduced antiviral activity, whereas the presence of aspartic acid at positions Ser28, Thr151, or Thr343 resulted in enhanced activity: referred to as hypermorphic mutants. In some cases, these hypermorphic mutations, particularly when paired with other MX2 mutations (e.g., S28D/T151D or T151D/T343A) acquired the capacity to inhibit HIV-1 capsid mutants known to be insensitive to wild-type MX2, such as P90A or T210K, as well as MX2-resistant retroviruses such as equine infectious anemia virus (EIAV) and murine leukemia virus (MLV). This work highlights the complexity and importance of MX2 phosphorylation in the regulation of antiviral activity and in the selection of susceptible viral substrates. IMPORTANCE Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. We recently demonstrated how phosphorylation of a stretch of three serines in the amino-terminal domain of MX2 inhibits the antiviral activity. Here, we identify additional positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensitive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses.

Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.

MX2 mediates establishment of interferon response profile, regulates XAF1, and can sensitize melanoma cells to targeted therapy.

MX2 is an interferon inducible gene that is mostly known for its antiviral activity. We have previously demonstrated that MX2 is also associated with the tumorigenesis process in melanoma. However, it remains unknown which molecular mechanisms are regulated by MX2 in response to interferon signaling in this disease. Here, we report that MX2 is necessary for the establishment of an interferon-induced transcriptional profile partially through regulation of STAT1 phosphorylation and other interferon-related downstream factors, including proapoptotic tumor suppressor XAF1. MX2 and XAF1 expression tightly correlate in both cultured melanoma cell lines and in patient-derived primary and metastatic tumors, where they also are significantly related with survival. MX2 mediates IFN growth-inhibitory signals in both XAF1 dependent and independent ways and in a cell type and context-dependent manner. Higher MX2 expression renders melanoma cells more sensitive to targeted therapy drugs such as vemurafenib and trametinib; however, this effect is XAF1 independent. In summary, we uncovered a new mechanism in the complex regulation of interferon signaling in melanoma that can influence both survival and response to therapy.

Effect of types of breeding on embryo survival following first AI in lactating Holstein cows.

The main objective was to investigate the effects of timed-AI protocols versus AI following oestrus detection on circulating progesterone (P4) and embryo survival after first service in Holstein cows. Cycling status was determined by ultrasonography and by plasma P4 concentrations 14 and 26 days after calving, and only cows with a corpus luteum and/or P4 ≥ 1 ng/ml were used. Cows were randomly allocated to one of three types of breeding: DO (n = 80), received GnRH-7d-PGF2α-3d-GnRH and Ovsynch56 was initiated 7 days later; G7G (n = 70), received PGF2α-2d-GnRH and Ovsynch56 (GnRH-7d-PGF2α-56h-GnRH-16h-AI) was initiated 7 days later; or AI based on oestrus detection, EDAI (n = 60). Progesterone was also determined at AI and 8, 16, 18 and 20 days after AI; ISG15 and MX2 mRNA abundance were determined 16 days after AI. Mean plasma P4 at AI was greater in the EDAI group compared with DO and G7G groups, while after AI, P4 was greater in DO and G7G groups compared with EDAI group. However, the percentage of cows with a concentration of P4 < 0.8 ng/ml at AI did not differ among groups. Relative mRNA abundance of ISG15 and MX2 was greater in the DO and G7G groups compared to those in EDAI group. Pregnancy per AI 16, 32 and 60 days after AI was greater (p < .05) in cows in the DO group compared with those in EDAI group (47.5%, 38.8% and 36.3% vs. 30.0%, 21.7% and 15.0%). Pregnancy losses between 16 and 60 days after AI were greater (p < .05) in cows in the EDAI (50.0%) group compared to those subjected to DO (23.7%) or G7G (24.1%). In conclusion, the use of timed-AI synchronization protocols resulted in greater circulating P4 concentrations post-AI and greater embryo survival following first service in lactating Holstein cows.

A Potent Postentry Restriction to Primate Lentiviruses in a Yinpterochiropteran Bat.

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.

Pro-515 of the dynamin-like GTPase MxB contributes to HIV-1 inhibition by regulating MxB oligomerization and binding to HIV-1 capsid.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

MX 2 is a novel regulator of cell cycle in melanoma cells.

MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.