PLOS Pathogens

3-nucleotidases/nucleases, distinct class I nucleases of protozoan parasites, play a pivotal role in extracellular purine salvage. As Leishmania are purine auxotrophs and lack de novo synthesis, ectoenzymes facilitating nucleotide and nucleic acid cleavage are indispensable for subsequent uptake. Employing quantitative proteomics, we identified a class I nuclease p1/s1 cluster in L. major that comprises enzymes exhibiting dual 3-nucleotidase and endonuclease activity. Expression of these enzymes is induced upon miltefosine or staurosporine treatment and was specifically detected in stationary-phase, but not in logarithmic-phase promastigotes. After confirming secretion of p1/s1, ecto-enzymatic activity was detected on parasites and in the culture supernatant. Viable null mutants deficient for the p1/s1 cluster were only obtained when a diCre-based inducible knockout system was applied, whereas direct deletion approaches were lethal. The viable knockout strains exhibited significantly reduced 3-nucleotidase/nuclease activity. Notably, these parasites adapted by compensatory enrichment of various alternative purine salvage proteins at the proteomic level. Furthermore, both enzymatic functions implied mechanisms of host-pathogen interactions to facilitate infection establishment: Utilizing 3-nucleotidase activity, Leishmania generate extracellular adenosine to suppress inflammatory cytokine secretion from macrophages and reduce lymphocyte proliferation in a human primary cell model. The presence of ecto-nucleases also allowed these parasites to degrade and survive neutrophil extracellular traps, a potent first-line innate immune mechanism in pathogen defense. In summary, our integrative approach combining proteomics, immunological and genome editing methods expands current knowledge about Leishmania major 3-nucleotidases/nucleases. By offering new insights into the diverse involvements in host-pathogen interactions, we highlight p1/s1 as pivotal factor during infection and potential drug target.

Toxoplasma gondii infection activates pattern recognition receptor (PRR) pathways that drive innate inflammatory responses to control infection. Necroptosis is a pro-inflammatory cell death pathway apart of the innate immune response that has evolved to control pathogenic infection. In this study we further defined the role of Z-DNA binding protein 1 (ZBP1) as a PRR and assessed its contribution to necroptosis as a host protection mechanism to T. gondii infection. We found that ZBP1 does not induce pro-inflammatory necroptosis cell death and ZBP1 null mice have reduced survival after oral T. gondii infection. In contrast, mice deleted in receptor-interacting serine/threonine-protein kinase 3 (RIPK3-/-), a central mediator of necroptosis, have significantly improved survival after oral T. gondii infection even with higher parasite burden. The physiological consequences of RIPK3 activity did not show any differences in intestine villi immunopathology but RIPK3-/- mice showed higher immune cell infiltration and edema in the lamina propria. The contribution of necroptosis to host survival was clarified with mixed lineage kinase domain like pseudokinase null (MLKL-/-) mice. We found MLKL-/- mice to succumb to oral T. gondii infection the same as wild type mice, indicating necroptosis-independent RIPK3 activity impacts host survival. These results provide new insights on the impacts of pro-inflammatory cell death pathways as a mechanism of host defense to oral T. gondii infection.

Jamestown Canyon Virus (JCV), a negative-sense arbovirus, is increasingly common in the upper Midwest of the USA. Transmitted by a range of mosquito genera, JCV has at its primary amplifying host, white-tailed deer. Aedes aegypti is the major transmitter globally of the positive-sense viruses dengue (DENV), Zika, chikungunya, and Yellow Fever. Ae. aegyptis distribution, once confined to the tropics, is expanding, in part due to climate change. Wolbachia, an insect endosymbiont, limits the replication of co-infecting viruses inside insects. The release and spread of the symbiont into Ae. aegypti populations has been effective in reducing transmission of dengue and other viruses to humans. The mechanism of Wolbachia-mediated viral blocking in vectors is still poorly understood, however. Here we explored JCV infection potential in Ae. aegypti, the nature of the vectors immune response, and interactions with Wolbachia infection. We show that Ae. aegypti is highly competent for JCV, growing to substantial loads and rapidly reaching the saliva after an infectious blood meal. The mosquito immune system responds with strong induction of RNAi and JAK/STAT. Neither the direct effect of viral infection nor the energetic investment in immunity appears to affect mosquito longevity. Wolbachia infection blocked JCV only in the early stages of infection. Wolbachia-induced immunity was small compared to that of JCV, suggesting innate immune priming does not likely explain blocking. We propose two models to explain why Wolbachias blocking of negative-sense viruses like JCV may be less than that of positive-sense viruses, relating to the slowdown of host protein synthesis and the triggering of interferon-like factors like Vago. In conclusion, we highlight the risk for increased human disease with the predicted future overlap of Ae. aegypti and JCV ranges. We suggest that with moderate Wolbachia-mediated blocking and distinct biology, negative-sense viruses represent a fruitful comparator model to other viruses for understanding blocking mechanisms in mosquitoes. Author SummaryJamestown Canyon Virus (JCV), a newly emerging virus in North America, causes disease when it spills out of its wild mammal hosts into human populations via the bite of infected mosquitoes. We show that the mosquito Aedes aegypti, known for transmitting many viral pathogens to humans globally, and whose distribution is creeping northward in the USA toward regions where JCV is present, is likely able to transmit the virus. Wolbachia is an endosymbiotic bacterium being released in wild mosquito populations of mosquitoes because it limits the replication of human viruses inside the mosquito, limiting their transmission to humans. We show that Wolbachia has a limited ability to control the replication of JCV, which is likely because Wolbachia-induced antiviral response is quite weak, and unique aspects of negative-sense virus biology make them less susceptible to blocking. Our findings suggest that JCV may serve as a comparative model to positive-sense viruses like dengue in dissecting the mechanism of Wolbachia-mediated virus blocking. It also warns that shifting mosquito distributions, as expected under a changing climate, could bring JCV and Aedes mosquitoes into greater contact, potentially increasing the incidence of JCV in humans.

Factors influencing Kaposis sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Here, using our unique model of KSHV infection in tonsil lymphocytes, we investigate the influence of host cytokines on the establishment of KSHV infection in human B cells. Our data demonstrate that KSHV manipulates the host cytokine microenvironment during early infection and susceptibility is generally associated with downregulation of multiple cytokines. However, we show that IL-21 signaling promotes KSHV infection by promoting both plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. Our data reveal that this phenotype is dependent upon a specific milieu of T cells, that includes IL-21 producing Th17, Tc17 and CD8+ central memory T cells. These results suggest that IL-21 plays a significant role in the early stages of KSHV infection in the human immune system and that specific immunological states favor the initial establishment of KSHV infection by increasing infection in plasma cells.

Toxoplasma gondii (T.gondii) hijacks host immune cells as Trojan Horse, and the infected cells accelerated the parasites dissemination. During acute infection, T.gondii specificity crosses the blood-brain-barrier to enter the brain. This selective mode of parasite transmission may be associated with the directed migration of infected immune cells. Immune cells follow various environmental cues for directional migration. However, the effect of T.gondii infection on the recognition of mechanical cues by immune cells remains unknown. Here, we examined the adhesion and migration of T.gondii-infected dendritic cells (DCs) on high and low stiffness substrates. We found that T.gondii infection alters the durotaxis migration of DCs. Infected DC exhibited stronger adhesion and lower migration on low stiffness substrates. In contrast to uninfected DCs, infected DCs migrated towards the low stiffness environment. TgWIP and TgROP17 co-regulate the F-actin structure of DCs and are involved in the formation of abnormal F-actin filaments. Rearrangement of the F-actin structure resulting from T.gondii infection regulates DCs abnormal recognition response to the mechanical cues. Recognition of DCs to the mechanical signals is independent of {beta}2- integrin expression. Meanwhile, challenging DCs with T.gondii increased the phosphorylation of focal adhesion kinase (FAK). Treatment with a FAK inhibitor (VS- 6063) influences the recognition response of infected DCs. FAK inhibition in adoptively transferred infected DCs effectively prevents the dissemination of T.gondii to the brain. The data reveal that T.gondii infection inversely affects the durotaxis of DCs by altering the phosphorylation level of FAK and remodeling of F-actin structure. T.gondii utilizes the change in DCs durotaxis migration to accelerate the parasites crossing the blood-brain-barrier. Author SummaryImmune cells travel through blood vessels and lymph vessels to various tissues, and respond to different types of environmental cues. Cells sense the cues and transmit these information to the cytoskeletal which induce directed cell migration towards or away from these signals. T.gondii infection remodeling the cytoskeletal of DCs which may cause abnormalities in these cues transduction. We found that T.gondii infection induces the formation of abnormal F-actin filaments in DCs, TgWIP and TgROP17 co-regulate the DCs F-actin structure. T.gondii infection increased the phosphorylation of FAK in DCs and has no effect with DCs surface {beta}2-integrin expression. These reasons lead to alter the original durotaxis migration of DCs, and makes infected-DCs tend to stay in the low stiffness environment. Meanwhile, the recognition response of infected DC to mechanical signal determines the parasite rapid crossing the blood-brain-barrier.

Human papillomavirus 16 (HPV16) is a high-risk alphapapillomavirus that is associated with cancers of mucosal epithelia. The virus genome exists in cells as an episome but can integrate and overexpress the E6 and E7 viral oncogenes. In related high-risk family members HPV18 and HPV31, host proteins including CTCF, an insulator, and SMC1A, a component of the cohesion complex, are known to interact with the viral genome and alter transcriptional activity, splicing patterns and episome amplification. However, the roles of these two proteins during HPV16 infection has not yet been fully examined. Here, we show during differentiation of the episomal HPV16-containing W12 cell line that CTCF association increases with the virus genome at the known E2 binding site, whilst additional CTCF binding now occurs at the putative L2 binding site, with SMC1A association occurring unchanged here. While expression of virus late transcripts (E4^L1, L2, L1) is stimulated, early transcript levels decrease by 48 hours, with the exception of the E6*IV spliced transcript. Conversely, in undifferentiated, monolayer W12 cells, CTCF knockdown increases the level of all early transcripts, whereas E6*IV level increases. Additionally, CTCF ablation as well as SMC1A knockdown results in decreases to HPV16 genome copy number. Taken together, this supports the model that while CTCF and SMC1A have a role in HPV16 genome maintenance, CTCF plays a greater part in regulating HPV16 oncogene splicing and expression during the natural lifecycle of the virus, and may be involved in a reduced risk of cancer development during episomal HPV16 infections.

Highlight statementThe findings highlight that TCV manipulates 26S proteasome and autophagy pathways to obstruct antiviral RNA decay defenses and ultimately enhance its ability to infect host cells. RNA decay is a pervasive process in eukaryotic cells. Viruses utilize the host cells intracellular machinery to gain access to essential molecules and subcellular structures required for infection during the pathogenesis process. The study demonstrates that turnip crinkle virus (TCV) infection enhances the expression of Arabidopsis Dcp1 (AtDcp1), which negatively regulates the accumulation of TCV RNA, indicating its involvement in antiviral defense. Nevertheless, TCV circumvents the antiviral defense based on RNA decay, as indicated by the capsid protein (CP) of TCV stabilizing the known nonsense-mediated RNA decay targeted transcripts. In vivo, CP physically interacts with AtDcp1, promoting AtDcp1 degradation via ubiquitination and autophagy pathways. This is evidenced by the observation that the degradation is inhibited by both 26S proteasome and autophagy inhibitors. Furthermore, CP elevates the polyubiquitination of Dcp1-Flag and the quantity of pre-autophagosome or autophagosome structures. These data indicate that CP suppresses RNA decay by interacting with AtDcp1 and mediating its degradation through the 26S proteasome and autophagy pathways, effectively subduing antiviral RNA decay. This study uncovers a previously unidentified virulence strategy in the ongoing conflict between plants and viruses.

The basal complex (BC) of Toxoplasma gondii has an essential role in cell division but details on the mechanism are lacking. To promote insights in this process, reciprocal proximity based biotinylation was used to map the basal complex proteome. An assembled protein map was interrogated by spatiotemporal characterization of critical components as well as functionally by disrupting the expression of the components. Spatially, this revealed four proteins sub-complexes with distinct sub-structural BC localization. Temporally, several patterns were differentiated based on their first appearance and/or disappearance from the BC corresponding with different steps in BC development (initiation, expansion, constriction, maturation). We also identified a protein pre-ceding BC formation (BCC0) laid out in a 5-fold symmetry. This symmetry marks the apical annuli and site of alveolar suture formation. From here, it was determined that the apical cap is assembled in the apical direction, whereas the rest of the IMC expands in the basal direction, inspiring a new bi-directional daughter budding process. Furthermore, we discovered BCC4, an essential protein exclusively localizing to the BC during cell division. Although depletion of BCC4 did not prevent BC formation, it led to BC fragmentation at the mid-point of cell division. Based on these data, a model is presented wherein BCC4 and MORN1 stabilize each other and form a rubber band that implies an essential role for the BC in preventing the fraying of the basal end of the assembling daughter cytoskeleton scaffolds. Furthermore, one new component of the Myosin J and Centrin2 cluster was BCC1, a hypothetical protein whose depletion prevents the non-essential last step of BC constriction. Overall, the BC is a highly dynamic, multi-functional structure that is critical to the hierarchical assembly of the daughter parasites.

Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus. It is associated with Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. KSHV is known to interact with several different receptors, among them heparan sulfate proteoglycans, Eph family receptors, and integrins. We mutated the closely related rhesus monkey rhadinovirus in the known receptor interaction sites for Eph family and Plexin domain containing proteins and found it to still replicate on certain cells. This lytic virus was then used as a selection agent in a genome-wide CRISPR knockout screen, which identified TIM1 and NRP1 as host dependency factors. NRP1 is also host factor for the related Epstein-Barr virus and was recently reported to promote KSHV infection, which we confirm even if it functions with low efficiency on most cells and became functional only after ablation of the Eph receptor interaction. Further analysis through overexpression demonstrated that Tim-1 and the related Tim-4 are strong mediators of RRV and KSHV infection, in particular in the absence of other receptor interactions and even more pronounced for a KSHV mutant deleted in glycoprotein K8.1. Both Tim-1 and Tim-4 are heavily O-glycosylated phosphatidylserine (PS) receptors. For KSHV in particular, experiments with mutated Tim-1 and comparison to Ebola virus glycoprotein-driven entry indicate that the interaction with Tim-1 occurs through PS-binding by Tim-1 and suggest additional interaction in a PS-independent manner. The mucin-like domain of Tim-1 is required for optimal receptor function. The use of Tim proteins for entry is a novelty for herpesviruses and underscores the unique biology of KSHV and RRV.

Toxoplasma gondii is a widespread protozoan parasite that has significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood, yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remains largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with the epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. Using CUT&Tag methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCEToxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasites life cycle stage transitions is needed for the development of new therapies aimed to treat toxoplasmosis and transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled by infected cats.

Viruses need cells to replicate and, therefore, ways to counteract the hosts immune response. Mitochondria play central roles in mediating innate immunity, hence some viruses have developed mechanisms to alter mitochondrial functions. Herein we show that arenavirus nucleoprotein (NP) enters the mitochondria of infected cells and affects their morphological integrity. We initially demonstrate electron-dense inclusions within mitochondria of reptarenavirus infected cells and hypothesized that these represent viral NP. Software predictions then serve to identify a putative N-terminal mitochondrial targeting signal (MTS) in arenavirus NPs; however, comparisons of wild-type and N-terminus mutated NPs suggest MTS-independent mitochondrial entry. NP does not enter isolated mitochondria, indicating that translocation requires additional cellular factors or conditions. Immune electron microscopy finally confirms the presence of NP within the mitochondria both in vitro and in infected animals. We hypothesize that mitochondria targeting might complement the known interferon antagonist functions of NP or alter the cells metabolic state.

Human papillomaviruses (HPV) typically cause chronic infections by modulating homeostasis of infected basal cell to ensure persistence. Using FUCCI and cell-cell competition assays, we established the role of two common viral targets of low-risk and high-risk E6 proteins, E6AP and NHERF1, on four key components of epithelial homeostasis. These includes cell density, proliferation, commitment to differentiation and basal layer delamination. Our RNA sequencing results validated E6s effects on homeostasis and revealed similar transcriptional gene regulation of E6-expressing cells and E6AP-/- cells. For example, yes-associated protein (YAP) target genes were up-regulated by either E6 expression or E6AP depletion. This is also supported by YAP expression pattern in both monolayer cell culture and HPV-infected clinical tissues. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways have not been defined. We demonstrate that deletion of E6AP in keratinocytes delayed the onset of differentiation and the abundance of E6AP is reduced in HPV-infected tissue. This suggests that Alpha E6 regulates epithelium homeostasis by inhibiting E6APs activity, leading to alteration of multiple downstream pathways including YAP activation. Potential treatments can thus be developed to resolve the reservoir of HPV infection.

Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.

Peptidoglycan Recognition Proteins (PGRPs) are conserved pattern-recognition receptors that detect microbe-associated molecular patterns (MAMPs) and activate host immune responses. Compared to other dipterans, the tsetse fly (Glossina morsitans morsitans) genome encodes only five PGRPs-PGRP-LA, -LB, -LC, -SA, and -SB - far fewer than most dipterans, likely reflecting its sterile blood diet and streamlined microbiota. Here, we identify PGRP-LA as a critical regulator of peritrophic matrix (PM) integrity in the cardia (proventriculus), the tissue responsible for PM production. The PM is a chitinous sleeve-like barrier that separates the midgut epithelium from the ingested bloodmeal, supporting digestive homeostasis and infection resistance. We show that pgrp-la is prominently expressed in the cardia, transiently induced after a bloodmeal in newly eclosed flies, and reinduced following subsequent feedings, likely in response to blood-constituents or mechanical stretch. This induction is sustained during microbial exposure and prolonged in trypanosome-infected flies. RNAi-mediated reduction of pgrp-la significantly increased the prevalence of midgut trypanosome infections, indicating a protective role during early infection. PGRP-LA did not mediate infection resistance via canonical IMD pathway signaling, as its silencing did not affect antimicrobial peptide expression. Instead, PGRP-LA modulated the expression of PM-associated genes and gut barrier integrity. Silencing pgrp-la reduced PM structure, increased midgut weights and enhanced fly survival following oral challenge with entomopathogen Serratia marcescens, likely due to earlier epithelial immune responses through a compromised PM. Similar phenotypes were observed when flies were fed anti-PGRP-LA antibodies, supporting a structural role for PGRP-LA. In addition, soluble variant surface glycoproteins (sVSGs) from trypanosomes and knockdown of microRNA-275 (miR-275), also decreased pgrp-la expression, suggesting that PGRP-LA is part of a broader regulatory network, including the miR-275/Wingless signaling. Collectively, our results identify PGRP-LA as novel regulator of PM biogenesis and vector competence in tsetse, expanding the functional repertoire of PGRPs in insect gut barrier maintenance beyond canonical immune signaling pathways. Author SummaryInsect vectors such as tsetse flies can be infected with pathogens that cause devastating disease in mammals. To protect themselves insect vectors rely on pattern recognition receptors (PRRs) that detect pathogens and activate the production of antimicrobial peptides (AMPs). Physical barriers in the gut also play an important role in limiting infections. One such barrier is the peritrophic matrix (PM), a sleeve-like structure that lines the insect gut and separates the blood meal and its contents from the underlying cells. For trypanosome parasites, which cause sleeping sickness in humans, the PM is the first barrier they must traverse to colonize the tsetses gut. In this study, we identified a PRR, PGRP-LA, that, unlike related proteins in other insects that activate AMPs, regulates the integrity of the tsetses protective PM barrier. When PGRP-LA was disrupted, the gut barrier weakened, and flies became more susceptible to trypanosome infection. Our work highlights a previously unrecognized role for PGRP-LA in maintaining gut barrier integrity and suggest that targeting this pathway could be a strategy to help reduce parasite transmission.

Ascaris is one of the most widespread helminth infections of humans and pigs, leading to chronic morbidity in humans and considerable economic losses in pig farming. Additionally, pigs are an important reservoir for the zoonotic bacterial pathogen Salmonella, where pigs can serve as asymptomatic carriers. Here, we investigated the impact of an ongoing Ascaris infection on the immune response to Salmonella in pigs. We observed higher bacterial burdens in experimentally coinfected pigs compared to pigs infected with Salmonella alone. Ascaris-infected pigs exhibited numerous hallmarks of a type 2 immune response in organs impacted by larval migration, including increased Th2 cells, increased IL-4 production, eosinophilia, and increased expression of CD206, a marker for alternatively activated macrophages. While we observed only mild changes in frequencies of CD4+ Treg, Ascaris- infected pigs had increased frequencies of CD8+ Treg. We show that type 2 immune signals enhance susceptibility of macrophages to Salmonella infection in vitro. Furthermore, Ascaris impaired Salmonella-induced monocytosis and TNF- production by myeloid cells. Hence, our data demonstrate widespread immunomodulation during an acute Ascaris infection that facilitates the microbial spread into gut-associated lymphoid tissue in a Salmonella coinfection. ImportanceIn experimentally infected pigs we show that an ongoing infection with the parasitic worm Ascaris suum modulates host immunity to render pigs more susceptible to invading Salmonella. Both infections are widespread in pig production and the prevalence of Salmonella is high in endemic regions of human Ascariasis, indicating that this is a clinically meaningful coinfection. We observed a type 2 immune response to be induced during an Ascaris infection correlating with an increased susceptibility of pigs to the concurrent bacterial infection.

The Kaposis sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by multiple copies of 0.8 kb terminal repeat (TR) sequence. While TRs function in plasmid maintenance is well-established, TRs transcription regulatory roles have not been fully explored. Here, we show KSHV TR is a large transcription regulatory domain. A series of Cleavage Under Targets & Release Using Nuclease demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modifications were conserved in unique region of the KSHV genome among naturally infected cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TRs. In the Ori-Lyt region, the LANA complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and two K-Rta recruitment sites frank the nucleosome. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function with TR. KSHV TR is, therefore a regulatory domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body with TR. KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex. ImportanceEnhancers are a crucial regulator of differential gene expression programs. Enhancer is the cis-regulatory sequences that determine target genes spatiotemporal and quantitative expression. Here, we show that KSHV terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. KSHV enhancer is occupied by LANA and its interacting proteins, such as CHD4, and CHD4 is known to restrict enhancers to access promoters for activation. This study thus proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates lytic gene transcription.

Viruses have developed many different strategies to counteract immune responses, and Vaccinia virus (VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here we show that ISG15 participates in the control of VACV dissemination. Infection of Isg15-/- mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, comparing with Isg15+/+ cells. Transmission electron microscopy revealed accumulation of intracellular and a decrease in extracellular virus particles in the absence of Interferon Stimulated Gene 15 (ISG15), consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Last, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. Author SummaryViral infections are a constant battle between the virus and the host. While the hosts only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the hosts resources. Along millions of years of incessant encounters, Poxviruses have developed a unique strategy consisting in the production two specialized "troops": intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination. The mechanism by which the virus decides to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus whose infectivity and host range depend on the coordinated functions of its DNA-A and DNA-B components. Here, we investigated the replication, movement, and systemic infection capacity of ToLCNDV isolates from India (IN) and Spain (ES) and their pseudo-recombinants in tomato and Nicotiana benthamiana. In tomato, the IN isolate (A-IN/B-IN) systemically infected all plants, whereas the ES isolate (A-ES/B-ES) failed to do so. Pseudo-recombinant analyses revealed that DNA-B from the IN isolate complemented A-ES in trans, enabling systemic spread, while B-ES accumulated poorly and supported systemic infection only inefficiently. Although both A components replicated locally in tomato, DNA-A from the IN isolate was unable or only marginally able to infect systemically in the absence of DNA-B, demonstrating an essential contribution of DNA-B to long-distance movement. In N. benthamiana, both isolates established systemic infections even as monopartite viruses, though with substantially reduced viral titers and attenuated symptoms, indicating that host factors can partially compensate for the absence of DNA-B. Using a DsRed-tagged {Delta}CP mutant of ToLCNDV-ES, we further show that coat protein is required for systemic movement in N. benthamiana, despite the presence of DNA-B-encoded movement functions. Collectively, these results uncover striking host- and isolate-dependent differences in component compatibility and demonstrate that systemic infection by ToLCNDV-IN DNA-A in tomato requires DNA-B, while ToLCNDV-ES additionally depends on coat protein for efficient movement in N. benthamiana.

To survive and efficiently transit between plant and insect hosts, circulative plant viruses have evolved sophisticated strategies to exploit insect vector factors. Tomato yellow leaf curl virus (TYLCV) is transmitted by Bemisia tabaci through a circulative and replicative pathway. In insects, C20 oxylipin (eicosanoid) and C18 oxylipin (EpOME) antagonistically regulate antiviral responses. Upon TYLCV infection, the intestinal apoptosis of B. tabaci facilitated the viral multiplication. The apoptosis was suppressed by eicosanoid but induced by EpOME. EpOME treatment also upregulated other proviral factors, including defensin, PGRP, and cathepsins, while eicosanoid signaling exerted opposite effects. TYLCV infection suppressed eicosanoid biosynthetic enzymes but induced a cytochrome P450 gene involved in EpOME biosynthesis, consistent with elevated EpOME levels in the viruliferous B. tabaci detected by LC-MS/MS. Individual RNA interference treatments specific to each of the TYLCV genes in the viruliferous insects revealed that only silencing of the viral C2 gene abolished EpOME-mediated proviral effects. These findings uncover a lipid-mediated mechanism by which TYLCV enhances vector competence to promote transmission. IMPORTANCEVarious plant viruses depend on insect vectors for their horizontal transfer. Some of them exhibit a circulative and propagative transmission by multiplying the viral titers within the insects using the host machinery. Here is a fascinating manipulation of the host immunity by a plant virus, tomato yellow leaf curl virus (TYLCV), which uses the insect endocrine signals associated with immunity of its vector, Bemisia tabaci. Two types of oxylipins, eicosanoid and EpOME, antagonistically act to insect immunity, in which eicosanoid induces immune responses while EpOME, as an insect immune resolvin, suppresses them. TYLCV uses its C2 gene component as a virulent factor to induce the EpOME biosynthesis of B. tabaci. The elevated EpOME levels in the vector insect led to proviral responses by inducing intestinal apoptosis and selectively suppressing the immune-associated genes. These findings demonstrate the viral manipulation of the host endocrine signal for inducing proviral responses.

Toxoplasma gondiis propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with {Delta}gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon gamma (IFN-{gamma}) in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-{kappa}B complex. While GRA15 similarly regulates NF-{kappa}B, infection with {Delta}gra83/{Delta}gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. ImportanceToxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasites ability to efficiently invade and regulate components of the hosts infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasmas ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasites diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogens tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cells response to limit infection.