Discovery of a Novel Bat Gammaherpesvirus

Zoonosis is the leading cause of emerging infectious diseases. In a recent article, R. S. Shabman et al. (mSphere 1[1]:e00070-15, 2016, http://dx.doi.org/10.1128/mSphere.00070-15) report the identification of a novel gammaherpesvirus in a cell line derived from the microbat Myotis velifer incautus. This is the first report on a replicating, infectious gammaherpesvirus from bats. The new virus is named bat gammaherpesvirus 8 (BGHV8), also known as Myotis gammaherpesvirus 8, and is able to infect multiple cell lines, including those of human origin. Using next-generation sequencing technology, the authors constructed a full-length annotated genomic map of BGHV8. Phylogenetic analysis of several genes from BGHV8 revealed similarity to several mammalian gammaherpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV)

Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the human malignancy Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. KSHV establishes lytic infection of monocytes in vivo , which may represent an important cellular reservoir during KS disease progression. KS tumors consist of latently infected endothelial cells; however, lytic phase gene products are important for KS onset. Early KS lesion progression is driven by proinflammatory cytokines supplied by immune cell infiltrates including T cells and monocytes. KSHV-infected monocytes may supply the lytic viral products and the inflammatory milieu conducive to KS tumor progression. To establish successful infection, KSHV extensively modulates the host immune system. KSHV antigens activate both innate and adaptive immune responses including KSHV-specific T cells, but lifelong infection is still established. Programmed death ligand 1 (PD-L1) is a prosurvival cell surface protein that suppresses T-cell-mediated killing. PD-L1 is variably present on various tumor cells and is a targetable marker for cancer treatment. We show that KSHV infection of human monocytes increases PD-L1 expression and transcription in a dose-dependent manner. We also saw evidence of lytic gene expression in the KSHV-infected monocytes. Intact KSHV is needed for full PD-L1 response in human monocytes. KSHV induces a general proinflammatory cytokine milieu including interleukins 1α, 1β, and 6, which have been implicated in early KS lesion progression. KSHV-mediated PD-L1 increase may represent a novel mechanism of KSHV-mediated immune modulation to allow for virus survival and eventually malignant progression. IMPORTANCE KSHV is the etiologic agent of Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. Programmed death ligand 1 (PD-L1) is an immunosuppressive cell surface marker that inhibits T cell activation. We report that KSHV infection of primary human monocytes upregulates PD-L1 transcription and protein expression. Analysis of the cytokine and chemokine milieu following KSHV infection of monocytes revealed that KSHV induces interleukins 1α, 1β, and 6, all of which have been implicated in KS development. Our work has identified another potential immune evasion strategy for KSHV and a potential target for immunotherapy of KSHV-derived disease

Runaway Kaposi Sarcoma-associated herpesvirus replication correlates with systemic IL-10 levels

KSHV-associated inflammatory cytokine syndrome (KICS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). KICS is associated with high-level, systemic replication of KSHV. This study characterized the clinical and virologic features of a KICS patient over time. Additionally, it compared the cytokine profiles of the KICS case to Kaposi's sarcoma (KS) (n = 11) and non-KS (n = 6) cases. This KICS case presented with elevated levels of KSHV and IL-10, as expected. Surprisingly, this case did not have elevated levels of IL-6 or human immunodeficiency virus 1 (HIV-1). Nevertheless, treatment with anti-IL6 receptor antibody (tocilizumab) reduced KSHV viral load and IL-10. The KSHV genome sequence showed no significant changes over time, except in ORF24. Phylogenetic analysis established this isolate as belonging to KSHV clade A and closely related to other US isolates. These findings suggest IL-10 as potential biomarker and therapy target for KICS

A viral kinase mimics S6 kinase to enhance cell proliferation

Viruses usurp the host cell machinery to replicate, disseminate, and propagate themselves. Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a viral protein kinase (vPK) also known as ORF36. Using in silico modeling and biochemistry, we report that vPK/ORF36 displays limited homology to cellular S6 kinase B1 (S6KB1). Both kinases share overlapping substrates and can phosphorylate S6. However, unlike S6KB1, vPK augments S6 phosphorylation under conditions where mammalian target of rapamycin (mTOR) is inhibited. vPK modulates cellular proliferation and protein synthesis, augments anchorage independence, and enhances angiogenesis. Depletion of vPK/ORF36 during lytic replication inhibits the production of infectious virions, thereby underscoring the importance of this kinase during the KSHV life cycle. Our collective observations suggest that vPK may function as a constitutively active mimic of S6KB1

RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the innate immune response against many RNA viruses. We previously showed that RIG-I restricts Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation (J. A. West et al., J Virol 88:5778–5787, 2014, https://doi.org/10.1128/JVI.03226-13). In this study, we report that KSHV stimulates the RIG-I signaling pathway in a RNA polymerase (Pol) III-independent manner and subsequently induces type I interferon (IFN) responses. Knockdown or inhibition of RNA Pol III had no effect on beta interferon (IFN-β) induction by KSHV. By using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) approach, we identified multiple KSHV regions that give rise to RNA fragments binding to RIG-I, such as ORF810420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650. The sequence dissimilarity between these fragments suggests that RIG-I detects a particular structure rather than a specific sequence motif. Synthesized ORF810420-10496 RNA stimulated RIG-I-dependent but RNA Pol III-independent IFN-β signaling. In summary, several KSHV RNAs are sensed by RIG-I in a RNA Pol III-independent manner.Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Innate immune responses against viral infections, especially the induction of type I interferon, are critical for limiting the replication of viruses. Retinoic acid-inducible gene I (RIG-I), a cytosolic RNA helicase sensor, plays a significant role in the induction of type I interferon responses following viral infection. Here, we identified multiple RNA regions in KSHV as potential virus ligands that bind to RIG-I and stimulate RIG-I-dependent but RNA Pol III-independent IFN-β signaling. Our results expand the role of RIG-I by providing an example of a DNA virus activating a canonical RNA-sensing pathway

Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the human malignancy Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. KSHV establishes lytic infection of monocytes in vivo, which may represent an important cellular reservoir during KS disease progression. KS tumors consist of latently infected endothelial cells; however, lytic phase gene products are important for KS onset. Early KS lesion progression is driven by proinflammatory cytokines supplied by immune cell infiltrates including T cells and monocytes. KSHV-infected monocytes may supply the lytic viral products and the inflammatory milieu conducive to KS tumor progression. To establish successful infection, KSHV extensively modulates the host immune system. KSHV antigens activate both innate and adaptive immune responses including KSHV-specific T cells, but lifelong infection is still established. Programmed death ligand 1 (PD-L1) is a prosurvival cell surface protein that suppresses T-cell-mediated killing. PD-L1 is variably present on various tumor cells and is a targetable marker for cancer treatment. We show that KSHV infection of human monocytes increases PD-L1 expression and transcription in a dose-dependent manner. We also saw evidence of lytic gene expression in the KSHV-infected monocytes. Intact KSHV is needed for full PD-L1 response in human monocytes. KSHV induces a general proinflammatory cytokine milieu including interleukins 1α, 1β, and 6, which have been implicated in early KS lesion progression. KSHV-mediated PD-L1 increase may represent a novel mechanism of KSHV-mediated immune modulation to allow for virus survival and eventually malignant progression

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

<div><p>Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic <i>Pseudomonas aeruginosa</i> lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to <i>P. aeruginosa</i> mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy <i>in vitro</i>, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the <i>mucA</i> gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both <i>P. aeruginosa</i> and <i>Escherichia coli</i>. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.</p></div

Proposed model of LL-37 induced mutagenesis.

<p>At sub-inhibitory concentrations, LL-37 can penetrate <i>P. aeruginosa</i> cells and enter the bacterial cytosol, where LL-37 dimers then bind to DNA. DNA binding by LL-37 then promotes DinB-dependent replication, which potentiates mutations in <i>mucA</i> leading to mucoid conversion. Alginate overproducing bacteria are then protected from lethal concentrations of LL-37 and mucoid variants are selected for and persist in CF.</p

Non-oxidative PMN pathways promote mucoid conversion.

<p>The mucoid conversion frequency was determined upon treatment of PAO1<i>algD-cat</i> with PMN lysates or granule preparations (<b>A</b>), sub-inhibitory concentrations (0.25 µM) of LL-37, human beta defensin 1 and 2 (hBD1/2) and human neutrophil peptide 1 (HNP1) (<b>B</b>), or sputum isolated from CF patients (<b>C</b>). In <b>C</b>, sputum was immune-depleted with a monoclonal LL-37 antibody or mouse IgG₁ isotype control antibody. All experiments were performed in triplicate on four independent occasions. Values are mean +/− SEM. Statistical analysis was carried out comparing HBSS or 10 mM sodium phosphate buffer (pH 6.2) (SPB) to PMN component treated in <b>A</b> and <b>B</b> and anti-LL-37 to isotype control treated sputum in <b>C</b>, using an unpaired two-tailed Mann-Whitney test (<i>* p</i>≤0.05, **<i>p</i>≤0.001).</p

LL-37 DNA binding promotes mucoid conversion.

<p><b>A,</b> homology modeling of LL-37 bound to <i>B</i>-DNA was performed manually on the basis of the backbone atomic coordinates of the homologous protein, sterol regulatory element binding protein, bound to DNA. In <b>B</b>, amino acid sequences of native LL-37 and synthetic derivatives are represented and the putative DNA binding region is indicated in yellow. Red: positive residues, blue: negative residues. In <b>C</b>, the percent of DNA bound by LL-37 derivatives was calculated from electrophoretic mobility shift assays (representative images in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004083#ppat.1004083.s004" target="_blank">Figure S4</a>), where densitometry was performed on each image using ImageJ. <b>D</b> represents the mucoid conversion frequency after treatment with LL-37 derivatives. Values are mean +/− SEM. Experiments were performed in triplicate on three independent occasions and statistical analysis was carried out using an unpaired two-tailed student's <i>t</i>-test (<b>C</b>) or Mann-Whitney test (<b>D</b>). (<i>* p</i>≤0.05).</p