PLOS Pathogens

Long non-coding RNAs (lncRNAs) are a class of regulatory RNAs with lengths exceeding 200 nucleotides. The lncRNA PURPL (p53 upregulated regulator of p53 levels) is a p53-regulated gene. PURPL is an oncogenic lncRNA that represses p53 function; it is upregulated in many tumors and promotes cancer cell proliferation. Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of all forms of Kaposi sarcoma (KS) and is tightly associated with primary effusion lymphoma (PEL). We found upregulation of PURPL in both de novo and chronically KSHV-infected cells. We found that p73 (a p53 homolog) associates with the PURPL promoter and upregulates PURPL expression. Previously, it has been shown that KSHV-encoded latency-associated nuclear antigen (LANA) stabilizes p73. We found that ectopic expression of LANA increased both p73 protein levels and PURPL expression. p73 knockdown abrogates PURPL upregulation following KSHV infection. Antisense oligonucleotides (ASO) targeting PURPL reduced PEL cell proliferation and increased acetylated p53 levels. In contrast to p53, p73 is not repressed by PURPL RNA. In summary, our study reveals how an oncogenic virus can utilize p73 to upregulate the oncogenic lncRNA PURPL as an additional way to repress p53. Moreover, we propose another mechanism by which p73 may promote tumor growth.

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), an opportunistic pulmonary infection of immunocompromised patients that can be fatal, despite treatment with modern antifungal drugs. During infection, myeloid cells, such as neutrophils, monocytes, and monocyte-derived dendritic cells (Mo-DCs) are recruited to the lung and are critical for phagocytosing and killing A. fumigatus spores. Recent evidence has shown that intracellular crosstalk between these professional immune cells and other resident pulmonary cell types is required for an effective immune response to A. fumigatus, and that signaling via the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is part of this intracellular communication. In this study, we sought to determine a role for GM-CSF signaling on monocytes and their progeny. We harnessed a mouse model of deletion of GM-CSF receptor {beta} chain (encoded by Csf2rb) in CCR2-expressing cells. Using this model, we found that GM-CSF signaling is required on CCR2+ cells for killing of A. fumigatus and for host survival. This observation expands the importance of GM-CSF in host defense during A. fumigatus pulmonary infection and extends its role in pathways of intracellular crosstalk that mediate effective pulmonary antifungal immunity.

The intracellular bacterium Wolbachia pipientis alters the biology of its arthropod hosts in many ways, and can increase resistance to RNA viruses in both Drosophila and mosquitoes. Wolbachia-induced pathogen blocking has generated much interest because of its potential to restrict insect vector transmission of human diseases caused by RNA viruses. However, the molecular mechanisms by which Wolbachia affects host viral resistance are still mostly elusive. We used dilp2-3,5 mutant Drosophila, which are long-lived, but only in the presence of Wolbachia, to show that the presence of Wolbachia also increased the resistance of the mutant flies to infection with Drosophila C virus (DCV), relative both to mutants lacking Wolbachia and to wild type flies with and without Wolbachia. The insulin mutant flies had higher Wolbachia titres than wild type flies. By RNA-seq analysis of the Wolbachia transcriptome, we identified Wolbachia genes that were more strongly expressed in dilp2-3,5 mutant flies. Ankyrin-domain-containing proteins were among the most strongly up-regulated and they are predicted to be secreted effector proteins. To address their effect on host physiology, we generated 4 transgenic fly lines each with inducible expression of a different genes encoding ankyrin-domain-containing proteins. Expression of 3 of these did not cause obvious effects, but expression of WD0754 at high levels severely shortened fly survival. Interestingly, however, chronic low-level induction of WD0754 increased the resistance of the flies to DCV infection. Proteomics analysis showed a robust, tissue-specific, anti-viral response upon WD0754 induction, and identified the NFkB-like IMD pathway as a potential mediator of the antiviral activity of WD0754. Consistently, WD0754 expression activated Relish, a key downstream transcriptional mediator of IMD signalling, while loss-of Relish blocked the antiviral effect of WD0754. In summary, we identified a Wolbachia-derived ankyrin domain containing protein that modulates host immunity through the IMD pathway.

Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro. In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana, we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, L. major promastigote development in vitro does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei. These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species.

Porcine deltacoronavirus (PDCoV) is a globally circulating coronavirus of swine that causes severe enteric disease and mortality in neonatal piglets. Its broad host range and zoonotic potential raises concerns about continued spillover into human populations. However, the host factors that govern PDCoV infection remain largely unexplored. Identifying host factors involved in virus infection may uncover novel therapeutic targets for antiviral drug development or targets for gene editing to create resistant pigs. Here, we conducted a genome-wide CRISPR knockout (GeCKO) screen to identify host factors contributing to PDCoV infection in swine testicular (ST) cells. Small guide RNAs (sgRNAs) targeting the ANPEP gene, expressing the main receptor porcine aminopeptidase N for PDCoV, were enriched in PDCoV-resistant cells, demonstrating the validity of the screen. Moreover, we identified transmembrane protein 30A (TMEM30A), an accessory {beta}-subunit of the P4-ATPase flippase complex, as a host dependency factor for PDCoV infection along with several potential PDCoV-induced cell death regulators, including DET1, IFNAR1, RFWD2, STAT1 and TP53. We further demonstrated that PDCoV attachment and internalization into cells were significantly impaired in TMEM30A knockout ST cells, suggesting that TMEM30A is important in the early stages of the PDCoV life cycle. Because TMEM30A also contributes to SARS-CoV-2 replication, future studies are warranted to investigate its mechanistic role in the context of coronavirus infection.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis, with an estimated 20 million human infections annually. Its diverse host range presents a significant zoonotic threat, yet the molecular mechanisms underpinning HEV-host interactions remain poorly characterised. Here, we used transposon-based insertion screening of the HEV pORF1 polyprotein to identify a novel region within the viral PCP domain that permits HA-epitope insertion, enabling the first epitope tagging of this domain. Combined with V5 tagging of the hypervariable region (HVR), this approach enabled the generation of a replication-competent stable cell line expressing dual-tagged ORF1. Co-immunoprecipitation and TMT-based LC-MS proteomics revealed that pORF1 interacts with host proteins involved in endoplasmic reticulum-associated degradation (ERAD) and the 26S proteasome. Pharmacological inhibition demonstrated that both pathways are required for efficient HEV replication. Treatment with the FDA-approved proteasome inhibitor bortezomib altered pORF1 precursor abundance and led to loss of full-length pORF1 through enhanced proteolysis. Importantly, the kinetics of ORF1 degradation following proteasome inhibition correlated with immediate loss of HEV replication, demonstrating a functional link between pORF1 proteostasis and viral replication. Collectively, our findings identify the ERAD network as a previously unrecognised requirement for HEV replication and establish a role for the 26S proteasome in maintaining pORF1 proteostasis.

Chronic hepatitis B virus (HBV) infection persists under current antiviral therapy because covalently closed circular DNA (cccDNA) remains in the infected hepatocytes as a durable transcriptional template. Although type I interferon (IFN-I) can reduce HBV cccDNA gene expression, the host effectors and mechanisms that restrict cccDNA remain incompletely defined. Using a minicircle-based HBV cccDNA expressing a Gaussia luciferase reporter gene (mcHBV-GLuc), we investigated how promyelocytic leukemia protein (PML) suppresses HBV cccDNA activity. Multiple PML isoforms reduced cccDNA-driven reporter activity and HBsAg production, and HBx-deficient ({Delta}X) mcHBV cccDNA was more susceptible than wild-type cccDNA to PML-mediated inhibition. Interestingly, efficient antiviral activity required the major PML SUMOylation sites. Disruption of SMC5/6 function attenuated PML-mediated repression of HBx-deficient cccDNA, associated with reduced PML SUMOylation and PML nuclear-body organization. These findings identify PML as a SUMO-dependent inhibitor of HBV cccDNA activity and reveal a context-dependent functional relationship between PML and SMC5/6 that is modulated by HBx.

High-risk human papillomaviruses (HPVs) are the etiological agents of over 5% of cancers worldwide including those of the cervix and oropharynx. HPVs infect stratified epithelia and establish latent infections in basal cells but restrict productive replication or amplification to differentiated suprabasal cells. Despite the presence of viral genomes in most differentiated cells, only a subset of cells amplifies genomes as well as express late genes and these correspond to those that have re-entered G2/M, but the factors regulating this selectivity are unknown. To determine the signals that control the productive viral life cycle, single cell RNA seq was performed on cells that stably maintain high-risk HPV genomes following differentiation. Ten populations of undifferentiated and differentiating keratinocytes were identified, however, only one differentiated population had entered G2/M, expressed late genes and amplified viral genomes. One of the highly expressed replication factors in this population was the type II topoisomerase TOP2 while no other topoisomerases were similarly induced. TOP2 was found to bind to viral genomes, and acute depletion in differentiating cells blocked entry into G2/M, impaired genome amplification and abrogated late gene expression. Amplification also requires activation of DNA repair pathways through the induction of high levels of DNA breaks, and TOP2 accounted for more than half of the breaks present in differentiating cells. Increases in levels of TOP2 in differentiating cells were driven by the E7 oncoprotein acting through the transcription factor FOXM1, which also controls expression of G2/M factors suggesting an auto-regulatory loop. These studies identify TOP2 as a critical regulator of HPV genome amplification upon differentiation.

Severe dengue is characterized by systemic inflammation and vascular leakage. Recent evidence indicates that Toll-like receptor 2 (TLR2) supports early steps of dengue virus (DENV) infection in monocytes, whereas intracellular viral RNA sensing drives much of the subsequent inflammatory response. However, whether this entry-associated function of TLR2 differs among human monocyte subsets remains unknown. Here, we investigated how the TLR2 axis regulates DENV serotype 2 (DENV2) infection and inflammatory output in purified primary human monocytes and sorted monocyte subsets. Blocking TLR2 at the cell surface or targeting its intracellular TIR domain reduced virus-cell association, productive infection, and the release of inflammatory and antiviral mediators. Among TLR2-associated receptors, CD14 and, to a lesser extent, CD36 contributed to efficient infection, whereas TLR1, TLR6, and TLR10 were dispensable under the conditions tested. Notably, TLR2 blockade reduced viral production and cytokine release in CD16- classical monocytes but not in pooled CD16+ monocytes, identifying a subset-specific dependence on the TLR2 axis. TLR3 contributed to inflammatory and antiviral responses without affecting viral replication, consistent with post-entry recognition of viral RNA. Finally, blocking the TLR2 axis during monocyte infection prevented the loss of endothelial barrier integrity induced by monocyte-derived soluble mediators. Together, these findings identify a TLR2-associated entry route that preferentially supports productive DENV2 infection in classical monocytes and thereby enables RNA-sensor-dependent inflammatory responses that compromise endothelial function.

Coxiella burnetii ( Cb ), the causative agent of Q fever, replicates within host macrophages by modulating innate immune responses through its type IVB secretion system (T4SS). Host cells sense pathogens via Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), to recognize pathogen-associated molecular patterns (PAMPs) and initiate signaling pathways that drive pro-inflammatory cytokine and interferon responses. Toll-like receptor 3 (TLR3) triggers the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Interferon regulatory factors (IRFs) resulting in pro-inflammatory cytokine production and type I interferon (IFN-I) induction. Here we demonstrate that Cb requires T4SS to suppress TLR3-induced NF-{kappa}B and IRF transcriptional responses. Furthermore, RNA purified from both virulent and avirulent Cb is sufficient to activate TLR3, identifying pathogen RNA as a relevant PAMP in this context. Using a pulmonary infection model with virulent Cb, we found that TLR3/ Toll-Interleukin-1 Receptor Domain-Containing Adapter Protein Inducing Interferon Beta (TRIF)-dependent innate immune pathway inhibits cachexia. In contrast, signaling through the interferon-/{beta} receptor (IFNAR) restricts bacterial dissemination, indicating that these pathways play distinct but complementary roles in host defense. Mechanistically, Cb suppresses TLR3/TRIF signaling in a T4SS-dependent manner by preventing host TLR3 recruitment to the Coxiella-containing vacuole (CCV) and disrupting TRIF-TNF Receptor-Associated Factor 6 (TRAF6) interactions, thereby selectively inhibiting impairing NF-{kappa}B activation. Consistent with this, we identify five T4SS effector proteins that attenuate TLR3-induced NF-{kappa}B signaling, including CBU1292, which suppresses both NF-{kappa}B and IRF-dependent responses downstream of TLR3.

Defective interfering particles (DIPs) profoundly influence viral replication and host responses yet it remains difficult to quantify their activity routinely in the laboratory. Here, using lymphocytic choriomeningitis virus (LCMV) as a model mammarenavirus, we establish complementary approaches for measuring interfering activity of DIPs across a range of experimental scales. First, we optimized plaque- and focus-based interference assays that directly quantify DIP-mediated suppression of standard infectious virus replication through a modified neutralization assay. Second, in cell monolayers saturated with both DIPs and infectious particles, we identified a complete loss of expression of the viral glycoprotein and matrix protein, which are each encoded in the positive-sense orientation of the mammarenavirus genome. Notably, this loss of protein expression mirrors the inhibition of plaque formation traditionally seen in this high DIP setting. The observed DIP-mediated loss of glycoprotein expression is a surrogate marker of interference and was used to develop an alternative assay of DIP activity. This glycoprotein expression assay recapitulated interference detected in traditional high-titer plaque assays (i.e. loss of plaques when all cells are saturated with DIPs and infectious particles) while substantially increasing scalability. These approaches enable sensitive and scalable quantification of DIP activity for LCMV as well as Junin virus and expand our experimental toolkit for studying arenavirus population dynamics, pathogenesis and immunity, and may help advance DIP-based antiviral strategies. This study also provides mechanistic details on how mammarenavirus DIPs interfere with infectious virus propagation.

Alphaviruses are mosquito-transmitted pathogens in the Togaviridae family that cause severe arthritogenic or encephalitic disease in humans. Swelling of peripheral joints and soft tissues is a hallmark of arthritogenic alphavirus infection, although the mechanism by which tissue edema occurs is not well understood. The cell surface protein, matrix remodeling-associated protein 8 (MXRA8), is a cell entry receptor for several arthritogenic alphaviruses. Global loss of MXRA8 expression in mice abrogates foot swelling and reduces virus dissemination. Here, we demonstrate that specific ablation of MXRA8 from platelet-derived growth factor receptor alpha (PDGFR) cells, including fibroblasts, phenocopies the foot swelling and dissemination deficits observed in chikungunya virus (CHIKV)-infected global MXRA8 knock-out mice. Edema in the foot requires CHIKV infection of PDGFR+ cells, which enhances both infection efficiency and permeability of endothelial cells. CHIKV-induced vascular leakage requires both MXRA8 expression and vascular endothelial growth factor signaling and contributes to dissemination by enabling virus access to the bloodstream. Collectively, our study establishes the role of MXRA8-dependent cell tropism in CHIKV dissemination and pathogenesis.

Epstein-Barr virus (EBV) infects 90% of the population worldwide and is associated with multiple malignancies, including Burkitt, Hodgkin and post-transplant lymphomas, nasopharyngeal and gastric carcinoma. EBV also triggers autoimmune diseases, including multiple sclerosis and systemic lupus erythematosus. While host factors important for EBV B-cell entry have been defined, including CD21/CR2, MHC class II and R9AP, much remains to be learned about host factors that support post-entry steps of EBV infection. To gain insight, we conducted a human genome-wide CRISPR-Cas9 screen. In addition to known EBV B cell receptors/co-receptors, we identified multiple new factors and pathways essential for EBV B cell infection. These included multiple B cell signaling pathways, actin cytoskeleton and nuclear import factors. Knockout of CD19, its tetraspanin chaperone CD81 or karyopherin subunit alpha 1 (KPNA1, also called importin-5) significantly impaired establishment of EBV infection. EBV and CD19 co-localized at early stages of EBV infection, and the CD19 cytoplasmic tail was important for EBV infection, suggesting CD19 signaling supports EBV uptake. Depletion of KPNA1 significantly impaired establishment of EBV infection, but did not impair production of infectious EBV upon reactivation of latently infected B cells. Together, these results provide insights into pathways that support EBV B cell infection and identify potential therapeutic targets.

Patients with severe dengue virus (DENV) infection can experience hemorrhage, shock and organ damage that coincides with defervescence and declining viremia. In addition to vascular leak, published work has shown that gut barrier permeability and elevated serum lipopolysaccharide (LPS) levels correlate with DENV disease severity. We previously described profound gut pathology in a mouse model of infection. This led to the hypothesis that a loss of gut epithelial barrier integrity and the influx of microbial products contribute to exacerbated inflammation and severe dengue disease. Consistent with this, here we demonstrated that DENV2 infection of AG129 mice compromised the gut epithelial barrier, promoted bacterial translocation, and induced microbial changes indicative of dysbiosis, including a reduced abundance of putatively beneficial microbial taxa and disrupted microbial circadian oscillations. Depleting gut bacteria with antibiotics from 1-day post-infection sharply reduced gut pathology as well as viral RNA in the proximal colon and liver, and the number of infected Kupffer cells in the liver. There was no evidence for systemic suppression of viral replication. We propose that viral infection of intestinal macrophages leads to inflammatory damage and mucosal barrier breakdown, dysbiosis and gut leak, with gut bacteria-dependent products promoting the susceptibility of Kupffer cells to DENV infection. Given the importance of liver damage in severe and fatal dengue disease, therapies that protect the gut barrier may help reduce disease severity.

Protein phosphatase 2A regulates eukaryotic signaling, but the function of its associated regulators remains poorly understood in the human fungal pathogen Cryptococcus neoformans. Here, we used affinity purification coupled with mass spectrometry and identified 81 candidate proteins associated with the catalytic subunit Pph22, including the scaffold subunit Tpd3, the regulator Tap42, and the protein phosphatase methylesterase 1. Conditional-expression and meiotic spore analyses supported an essential role for TAP42 in viability. We identified CNAG_05505 as the Rrd2 ortholog and detected an interaction between Rrd2 and Pph22 in a yeast two-hybrid assay. Deletion of RRD2 impaired thermotolerance, altered responses to multiple stresses and antifungal agents, reduced melanin and capsule production, delayed sexual development, and inhibited titan cell formation. Mutants also displayed altered cell wall staining and reduced expression of genes involved in chitin synthesis and deacetylation. RRD2 deletion increased basal Hog1 phosphorylation, whereas PPH22 overexpression reduced it. Double-mutant analysis revealed phenotype-dependent genetic interactions between RRD2 and HOG1. Although RRD2 deletion did not significantly alter overall survival in the Drosophila infection assay, it markedly attenuated virulence in mice: all mutant-infected mice survived the 70-day observation period, and fungal burdens were reduced across multiple organs at 21 days post-infection. These findings distinguish the essential contribution of Tap42 from the broad roles of Rrd2 in stress adaptation, morphogenesis, cell surface organization, and mammalian virulence, extending the functional analysis of the cryptococcal protein phosphatase 2A regulatory network.

Peach shoot blight (PSB), caused by Diaporthe amygdali, is one of the most devastating fungal diseases affecting peach production worldwide. Glutaredoxins (Grxs) are ubiquitous redox-regulatory proteins involved in antioxidant defense and iron metabolism, yet their functions in phytopathogenic fungi remain largely unknown. Here, we identified four Grxs (DaGrxA-DaGrxD) in D. amygdali. Among them, DaGrxB, a monothiol glutaredoxin, is the sole member essential for vegetative growth, asexual reproduction and virulence. DaGrxB interacts with iron-regulatory transcription factors DaHapX and DaSreA and modulates their activities via iron-dependent nucleocytoplasmic shuttling, coordinating iron acquisition and storage gene expression. During infection, D. amygdali encounters a biphasic iron environment: an inherently iron-limited host environment at early stages (1-2 dpi) followed by host-mediated iron excess (3-4 dpi), and DaGrxB is essential for navigating this transition. Consistently, deletion of either DaHAPX or DaSREA significantly reduced virulence, confirming that iron homeostasis is a critical determinant of D. amygdali virulence. Notably, deletion of DaGRXB disrupts Fe-S cluster biosynthesis, causing mitochondrial Fe2+ accumulation, impaired aconitase activity, and reduced ATP production, which triggers constitutive activation of the GSH-dependent antioxidant system, with elevated GSH/GSSG ratios, increased GR and GST activities, and reduced H2O2 levels. Exogenous GSH and H2O2 depletion both reduced virulence, suggesting that disturbed redox balance directly contributes to attenuated virulence. Additionally, the Trx domain assists the Grx domain in nucleocytoplasmic translocation through intramolecular interactions. Finally, LDH-delivered dsRNA targeting DaGRXB effectively controls PSB. Collectively, these findings reveal how a monothiol glutaredoxin integrates iron homeostasis, redox balance and virulence in a plant pathogen, providing a foundation for RNAi-based disease management.

Serious pertussis infections disproportionately affect infants but the biological basis for this age-dependent susceptibility remains unclear. Infant mouse models recapitulate features of severe human infant pertussis. We investigated the role of interferon lambda (IFN-{lambda}), a key regulator of mucosal immunity, in Bordetella pertussis infection of infant mice. While infected adult mice upregulate lung IFN-{lambda}, infant mice inoculated at P7 fail to upregulate IFN-{lambda} and succumb to infection. We hypothesized that failure to produce IFN-{lambda} during infection represents a critical immunological deficit in infant mice, and that restoring IFN-{lambda} signaling would improve survival outcomes. Whereas wild-type mice gained complete protection from lethal B. pertussis infection by P10, mice lacking the IFN-{lambda} receptor component IFNLR1 did not achieve full protection until P21. Loss of IFNLR1 was associated with enhanced bacterial dissemination to systemic organs. Infant mice possessed a functional IFN-{lambda} receptor in the lungs but failed to upregulate IFN-{lambda} during infection, and supplementing IFN-{lambda} exogenously extended survival. RNA sequencing of lung tissue from infected and uninfected wild-type and IFNLR1-deficient mice inoculated at different ages identified an immune transcriptional framework distinguishing susceptible from resistant animals at a systems level and revealed IFN-{lambda} signaling as a critical driver of immunological maturation in the infant lung. Infected infant IFNLR1-deficient mice had dysregulated immune cell recruitment to the lungs, indicating a quantitatively expanded but qualitatively impaired response. These findings demonstrate that IFN-{lambda} affects immune maturation accounting for a critical window of age-dependent resistance to lethal pertussis with novel therapeutic possibilities for human infants with this disease.

Epigenetic processes play important roles in the biology of the malaria parasite Plasmodium falciparum. We recently showed that Plasmodium histones are not only acetylated and methylated but also lactylated. This new epigenetic mark could form a particularly important axis of host-parasite signalling because severe malaria is often characterised by hyperlactataemia. In the human host this can cause respiratory distress, which is potentially fatal. For the parasite, it could be advantageous to sense this state of pathology and respond by modulating virulence. Virulence processes known to be under epigenetic control include antigenic variation, invasion switching and conversion to sexual gametocytes, i.e. gametocytogenesis. Here, we investigated the influence of lactate on gametocytogenesis. Hyperlactataemia in malaria patients is defined as [≥] 5mM blood lactate and we confirmed that in laboratory culture, adding 5mM L-lactate was sufficient to boost gametocytogenesis. Expression of the GDV-1 gene increased within one cell cycle of asexual parasites being exposed to L-lactate, suggesting that the canonical epigenetic switch for gametocytogenesis was involved: GDV-1 regulates epigenetic de-repression of the gene encoding the master transcription factor for gametocytogenesis, AP2-G. Indeed, parasite histones became rapidly lactylated after L-lactate exposure and chromatin profiling by CUT&Tag detected inducible lactylation of chromatin specifically upstream of the AP2-G gene, and also of the antisense RNA that regulates GDV-1. Thus L-lactate joins S-adenosylmethionine as a metabolite that can epigenetically regulate gametocytogenesis in P. falciparum.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. Common marmosets, a potential non-human primate model for vaccine development and infection studies, have previously been shown to support KSHV infection. We established infection of marmosets with BAC-derived, recombinant KSHV to analyze the role of individual glycoproteins (GPs) in tissue tropism. Three groups of four animals were infected intravenously with BAC-derived KSHV wild type (wt), EphA2-receptor-binding-deficient KSHV gHASAELAAN, or KSHV{triangleup}K8.1, lacking glycoprotein K8.1. Viral DNA was detected in multiple tissues of all animals, with the highest loads in spleen, liver, heart, and bone marrow. In most tissues, viral DNA loads did not significantly differ between groups. However, KSHV{triangleup}K8.1-infected animals exhibited significantly lower KSHV DNA levels in liver and bone marrow. KSHV wt infection induced activation of CD4+ and CD8+ T-cells. In animals infected with KSHV wt, KSHV gHASAELAAN or KSHV{triangleup}K8.1, different B cell subpopulations expanded after infection, which suggests that the glycoproteins themselves or the cell tropism imparted by their presence or absence might shape the host response to KSHV. In summary, our findings demonstrate that neither the Eph receptor interaction nor GP K8.1 is essential for intravenous infection, but K8.1 critically contributes to KSHV tissue tropism in vivo.

Herpes simplex virus-1 (HSV-1) has infected more than 67% of the world population causing diseases that range in severity from benign cold sores to encephalitis. To infect and persist in the host, HSV-1 has evolved sophisticated strategies to counteract antiviral responses. The viral immediate early protein ICP0 (Infected Cells Protein 0) plays a fundamental role in this process. ICP0 is an E3 ubiquitin ligase that is required for successful onset of lytic infection and productive reactivation of viral genomes from latency. Most ICP0 studies have been focused on its nuclear functions, but late gene expression drives accumulation of ICP0 to the cytoplasm where its functions are unknown. We recently demonstrated that cytoplasmic ICP0 localizes on the surface of endosomes through its interaction with CIN85 (Cbl-interacting protein of 85 kDa), a scaffolding protein for factors involved in endocytosis. This interaction is required for extrusion of host antiviral factors and immunoevasion. Here, we report that the E3 ubiquitin ligase activity of ICP0 is also required for these effects, as disruption of ICP0 RING finger domain, which disrupts its catalytic activity, resulted in an increase in the number of CIN85 endosomes that cannot be exocytosed. These endosomes colocalize with autophagosome components, but they do not enter the lysosomal pathway for degradation. Mutant viruses that cannot exocytose CIN85 endosomes produce EVs that are more potent at activating antiviral responses.