mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host's intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii. Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

ER-Phagy and Microbial Infection

The endoplasmic reticulum (ER) is an essential organelle in cells that synthesizes, folds and modifies membrane and secretory proteins. It has a crucial role in cell survival and growth, thus requiring strict control of its quality and homeostasis. Autophagy of the ER fragments, termed ER-phagy or reticulophagy, is an essential mechanism responsible for ER quality control. It transports stress-damaged ER fragments as cargo into the lysosome for degradation to eliminate unfolded or misfolded protein aggregates and membrane lipids. ER-phagy can also function as a host defense mechanism when pathogens infect cells, and its deficiency facilitates viral infection. This review briefly describes the process and regulatory mechanisms of ER-phagy, and its function in host anti-microbial defense during infection.

HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

As a fierce pathogen, spring viremia of carp virus (SVCV) can cause high mortality in the common carp, and its glycoprotein (G protein) is a component of the viral structure on the surface of virion, which is crucial in viral life cycle. This report adopted tandem affinity purification (TAP), mass spectrometry analysis (LC-MS/MS), immunoprecipitation, and confocal microscopy assays to identify Heat shock cognate protein 70 (HSC70) as an interaction partner of SVCV G protein. It was found that HSC70 overexpression dramatically inhibited SVCV replication, whereas its loss of functions elicited opposing effects on SVCV replication. Mechanistic studies indicate that HSC70 induces lysosomal degradation of ubiquitinated-SVCV G protein. This study further demonstrates that Membrane-associated RING-CH 8 (MARCH8), an E3 ubiquitin ligase, is critical for SVCV G protein ubiquitylation and leads to its lysosomal degradation. Furthermore, the MARCH8 mediated ubiquitylation of SVCV G protein required the participation of HSC70 through forming a multicomponent complex. Taken together, these results demonstrate that HSC70 serves as a scaffold for MARCH8 and SVCV G, which leads to the ubiquitylation and degradation of SVCV G protein and thus inhibits viral replication. These findings have established a novel host defense mechanism against SVCV.

Convergent Loss of the Necroptosis Pathway in Disparate Mammalian Lineages Shapes Viruses Countermeasures

Programmed cell death is a vital process in the life cycle of organisms. Necroptosis, an evolutionary form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages, central components of necroptosis, such as RIPK3 and MLKL, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores’ genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the N-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.

The IL-33-ILC2 pathway protects from amebic colitis

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2–/– mice but not RAG2−/−γc−/− mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2−/− mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2−/−γc−/− mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Detection of SARS-CoV-2 Derived Small RNAs and Changes in Circulating Small RNAs Associated with COVID-19

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3’-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.

The IL-33-ILC2 pathway protects from amebic colitis

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage during infection. Treatment with recombinant IL-33 protected mice from amebic infection and colonic tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to infection and weight loss. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the colon. Type 2 immune responses, which are known to be involved in tissue repair, were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33 mediated protection occurred in the absence of T or B cells but required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33 mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

MARCO+ lymphatic endothelial cells sequester arboviruses to limit viremia and viral dissemination

While viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the dLN and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. These findings uncover a previously unrecognized arbovirus scavenging role for LECs and improve our mechanistic understanding of viremia control during arboviral infections.

FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID 19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.

Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection

SUMMARYSARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high-molecular weight glycoproteins, as a prominent viral restriction network. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.