Host Cell Innate Immune Response Research Articles

Host Cells Upregulate Phosphate Transporter PIT1 to Inhibit Ehrlichia chaffeensis Intracellular Growth.

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Activation of protein kinase receptor (PKR) plays a pro-viral role in mammarenavirus-infected cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.

Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-β. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts.

Role of metapneumoviral glycoproteins in the evasion of the host cell innate immune response.

Human metapneumovirus (HMPV), an unsegmented negative-strand RNA virus, is the second most detected respiratory pathogen and one of the leading causes of respiratory illness in infants and immunodeficient individuals. HMPV infection of permissive cells in culture triggers a transient IFN response, which is efficiently suppressed later in infection. We report that two structural glycoproteins of the virus - namely G (Glycoprotein) and SH (Small Hydrophobic) - suppress the type I interferon (IFN) response in cell culture. This is manifested by inhibition of diverse steps of IFN induction and response, such as phosphorylation and nuclear translocation of IFN regulatory factor-3 and -7 (IRF3, IRF7), major transcription factors of the IFN gene. Furthermore, HMPV suppresses the cellular response to IFN by inhibiting the phosphorylation of STAT1 (Signal Transducer and Activator of Transcription 1), required for the induction of IFN-stimulated genes that act as antivirals. Site-directed mutagenesis revealed an important role of critical cysteine (Cys) residues in the Cys-rich carboxy terminal region of the SH protein in IFN suppression, whereas for G, the ectodomain plays a role. These results shed light on the mechanism of IFN suppression by HMPV, and may also offer avenues for new antiviral approaches in the future.

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.

RNA-Seq based transcriptome analysis during bovine viral diarrhoea virus (BVDV) infection

BackgroundBovine viral diarrhoea virus (BVDV) is the member of the genus Pestivirus within the Flaviviridae family and responsible for severe economic losses in the cattle industry. BVDV can employ ‘infect-and-persist’ strategy and ‘hit-and-run’ strategy to remain associated with hosts and thus contributes to BVDV circulation in cattle herds. BVDV have also evolved various strategies to evade the innate immunity of host. To further understand the mechanisms by which BVDV overcomes the host cell innate immune response and provide more clues for further understanding the BVDV-host interaction, in this descriptive study, we conducted a investigation of differentially expressed genes (DEGs) of the host during BVDV infection by RNA-Seq analysis.ResultsOur analysis identified 1297, 1732, 3072, and 1877 DEGs in the comparison groups mock vs. MDBK cells infected with BVDV post 2 h (MBV2h), mock vs. MBV6h, mock vs. MBV12h, and mock vs. MBV24h, respectively. The reproducibility and repeatability of the results were validated by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed the host DEGs that are potentially induced by BVDV infection and may participate in BVDV-host interactions. Protein-protein interaction (PPI) network analyses identified the potential interactions among the DEGs. Our findings suggested that BVDV infection induced the upregulation of genes involved in lipid metabolism. The expression of genes that have antiviral roles, including ISG15, Mx1, OSA1Y, were found to be downregulated and are thus potentially associated with the inhibition of host innate immune system during BVDV infection. The expression levels of F3, C1R, KNG1, CLU, C3, FB, SERPINA5, SERPINE1, C1S, F2RL2, and C2, which belong to the complement and coagulation signalling cascades, were downregulated during BVDV infection, which suggested that the complement system might play a crucial role during BVDV infection.ConclusionIn this descriptive study, our findings revealed the changes in the host transcriptome expression profile during BVDV infection and suggested that BVDV-infection induced altering the host’s metabolic network, the inhibition of the expression of antiviral proteins and genes within the complement system might be contributed to BVDV proliferation. The above findings provided unique insights for further studies on the mechanisms underlying BVDV-host interactions.

Transcriptome analysis of PK-15 cells in innate immune response to porcine deltacoronavirus infection.

Porcine deltacoronavirus (PDCoV) is a newly emerged swine enteropathogenic coronavirus affecting pigs of all ages and causing diarrhea problems. Research findings indicate that PDCoV has evolved strategies to escape innate immune response in host cells, but mechanism of PDCoV in innate immune modulation is not well understood. In this study, we report our findings on identifying the alterations of host cell innate immune response affected by PDCoV infection and exploring the gene expression profiles of PK-15 cells at 0, 24, and 36 h PDCoV post infection by RNA sequencing. A total of 3,762 and 560 differentially expressed genes (DEGs) were screened by comparison of uninfected PK-15 cells and infected PK-15 cells at 24 h post infection (hpi) (INF_24h versus NC), and also comparison of infected PK-15 cells between 24 and 36 hpi (INF_36h versus INF_24h), which included 156 and 23 porcine innate immune-related genes in the DEGs of INF_24h versus NC and INF_36h versus INF_24h, respectively. Gene Ontology function classification and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis were performed based on the DEGs that exhibited the same expression tendencies with most of the innate immune-associated genes among these PK-15 cell samples described above. The enrichment results indicated that extensive gene functions and signaling pathways including innate immune-associated functions and pathways were affected by PDCoV infection. Particularly, 4 of 5 innate immune signaling pathways, which were primarily affected by PDCoV, played important roles in I-IFN’s antiviral function in innate immune response. Additionally, 16 of the host cell endogenous miRNAs were predicted as potential contributors to the modulation of innate immune response affected by PDCoV. Our research findings indicated that the innate immune-associated genes and signaling pathways in PK-15 cells could be modified by the infection of PDCoV, which provides a fundamental foundation for further studies to better understand the mechanism of PDCoV infections, so as to effectively control and prevent PDCoV-induced swine diarrheal disease outbreaks.

The Cellular DExD/H-Box RNA Helicase UAP56 Co-localizes With the Influenza A Virus NS1 Protein.

UAP56, a member of the DExD/H-box RNA helicase family, is essential for pre-mRNA splicing and mRNA export in eukaryotic cells. In influenza A virus-infected cells, UAP56 mediates viral mRNA nuclear export, facilitates viral ribonucleoprotein complex formation through direct interaction with the viral nucleoprotein, and may indirectly affect antiviral host responses by binding to and/or facilitating the activation of the antiviral host factors MxA and PKR. Here, we demonstrate that UAP56 also co-localizes with the influenza A viral NS1 protein, which counteracts host cell innate immune responses stimulated by virus infection. The UAP56–NS1 association relies on the RNA-binding residues R38 and K41 in NS1 and may be mediated by single-stranded RNA. UAP56 association with NS1 does not affect the NS1-mediated downregulation of cellular innate immune pathways in reporter gene assays, leaving in question the exact biological role and relevance of the UAP56–NS1 association.

The Dengue Virus Replication Complex: From RNA Replication to Protein-Protein Interactions to Evasion of Innate Immunity.

Viruses from the Flavivirus family are the causative agents of dengue fever, Zika, Japanese encephalitis, West Nile encephalitis or Yellow fever and constitute major or emerging public health problems. A better understanding of the flavivirus replication cycle is likely to offer new opportunities for the design of antiviral therapies to treat severe conditions provoked by these viruses, but it should also help reveal fundamental biological mechanisms of the host cell. During virus replication, RNA synthesis is mediated by a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC). The RC is composed of both viral and host-cell proteins that assemble within vesicles composed of the endoplasmic reticulum membrane, near the nucleus. At the heart of the flavivirus RC lies NS4B, a viral integral membrane protein that plays a role in virulence and in down-regulating the innate immune response. NS4B binds to the NS2B-NS3 protease-helicase, which itself interacts with the NS5 methyl-transferase polymerase. We present an overview of recent structural and functional data that augment our understanding of how viral RNA is replicated by dengue virus. We focus on structural data that illuminate the various roles played by proteins NS2B-NS3, NS4B and NS5. By participating in viral RNA cap methylation, the NS5 methyltransferase enables the virus to escape the host cell innate immune response. We present the molecular basis for this activity. We summarize what we know about the network of interactions established by NS2B-NS3, NS4B and NS5 (their "interactome"). This leads to a working model that is captured in the form of a rather naïve "cartoon", which we hope will be refined towards an atomic model in the near future.

The nucleolar protein GLTSCR2 is required for efficient viral replication.

Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2) is a nucleolar protein. In the investigation of the role of GLTSCR2 that played in the cellular innate immune response to viral infection, we found GLTSCR2 supported viral replication of rhabdovirus, paramyxovirus, and coronavirus in cells. Viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm that enabled GLTSCR2 to attenuate type I interferon IFN-β and support viral replication. Cytoplasmic GLTSCR2 was able to interact with retinoic acid-inducible gene I (RIG-I) and the ubiquitin-specific protease 15 (USP15), and the triple interaction induced USP15 activity to remove K63-linked ubiquitination of RIG-I, leading to attenuation of RIG-I and IFN-β. Blocking cytoplasmic translocation of GLTSCR2, by deletion of its nuclear export sequence (NES), abrogated its ability to attenuate IFN-β and support viral replication. GLTSCR2-mediated attenuation of RIG-I and IFN-β led to alleviation of host cell innate immune response to viral infection. Our findings suggested that GLTSCR2 contributed to efficient viral replication, and GLTSCR2 should be considered as a potential target for therapeutic control of viral infection.

Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages.

Background Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections.Principal Findings In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype.ConclusionsThese findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.

Mutations within the conserved NS1 nuclear export signal lead to inhibition of influenza A virus replication.

BackgroundThe influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES).MethodsIn order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence.ResultsNS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Impaired activation of Akt phosphorylation by the replication-deficient viruses indicates possible disruption of NS1-p85β interaction by mutations in the NES region.ConclusionWe conclude that mutations within the NS1 NES result in impairment of several NS1 functions which extends further from the NES site being only involved in regulating the nuclear-cytoplasmic trafficking of NS1.

A model system for in vitro studies of bank vole borne viruses.

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2.In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.

Rotavirus Antagonism of the Innate Immune Response

Rotavirus is a primary cause of severe dehydrating gastroenteritis in infants and young children. The virus is sensitive to the antiviral effects triggered by the interferon (IFN)-signaling pathway, an important component of the host cell innate immune response. To counteract these effects, rotavirus encodes a nonstructural protein (NSP1) that induces the degradation of proteins involved in regulating IFN expression, such as members of the IFN regulatory factor (IRF) family. In some instances, NSP1 also subverts IFN expression by causing the degradation of a component of the E3 ubiquitin ligase complex responsible for activating NF-κB. By antagonizing multiple components of the IFN-induction pathway, NSP1 aids viral spread and contributes to rotavirus pathogenesis.

Human papillomaviruses: basic mechanisms of pathogenesis and oncogenicity

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the cutaneous and mucosal epithelium. Infection by specific HPV types has been linked to the development of cervical carcinoma. HPV infects epithelial cells that undergo terminal differentiation and so encode multiple mechanisms to override the normal regulation of differentiation to produce progeny virions. Two viral proteins, E6 and E7, alter cell cycle control and are the main arbitrators of HPV-induced oncogenesis. Recent data suggest that E6 and E7 also play a major role in the inhibition of the host cell innate immune response to HPV. The E1 and E2 proteins, in combination with various cellular factors, mediate viral replication. In addition, E2 has been implicated in both viral and cellular transcriptional control. Despite decades of research, the function of other viral proteins still remains unclear. While prophylactic vaccines to block genital HPV infection will soon be available, the widespread nature of HPV infection requires greater understanding of both the HPV life cycle as well as the mechanisms underlying HPV-induced carcinogenesis.