Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the β₁ integrin, the involvement of fibronectin and β₁ integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.

IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1β) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1β stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-B activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1β. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.

IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.

The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-vNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-vNr-13 showed similar growth kinetics; however, at early time points, HVT-vNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-vNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells.

IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek’s disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo. Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.

IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/β/, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4⁺ T lymphocytes and resting CD4⁺ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).

IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.

Macrophages in the lung detect and respond to influenza A virus (IAV), determining the nature of the immune response. Using terminal-depth cap analysis of gene expression (CAGE), we quantified transcriptional activity of both host and pathogen over a 24-h time course of IAV infection in primary human monocyte-derived macrophages (MDMs). This method allowed us to observe heterogenous host sequences incorporated into IAV mRNA, "snatched" 5' RNA caps, and corresponding RNA sequences from host RNAs. In order to determine whether cap-snatching is random or exhibits a bias, we systematically compared host sequences incorporated into viral mRNA ("snatched") against a complete survey of all background host RNA in the same cells, at the same time. Using a computational strategy designed to eliminate sources of bias due to read length, sequencing depth, and multimapping, we were able to quantify overrepresentation of host RNA features among the sequences that were snatched by IAV. We demonstrate biased snatching of numerous host RNAs, particularly small nuclear RNAs (snRNAs), and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then used a systems approach to describe the transcriptional landscape of the host response to IAV, observing many new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments.

IMPORTANCE Infection with influenza A virus (IAV) infection is responsible for an estimated 500,000 deaths and up to 5 million cases of severe respiratory illness each year. In this study, we looked at human primary immune cells (macrophages) infected with IAV. Our method allows us to look at both the host and the virus in parallel. We used these data to explore a process known as "cap-snatching," where IAV snatches a short nucleotide sequence from capped host RNA. This process was believed to be random. We demonstrate biased snatching of numerous host RNAs, including those associated with snRNA transcription, and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then describe the transcriptional landscape of the host response to IAV, observing new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments.

Viruses commonly antagonize innate immune pathways that are primarily driven by nuclear factor kappa B (NF-B), interferon regulatory factor (IRF), and the signal transducer and activator of transcription proteins (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased transcriptome sequencing (RNA-seq)-based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that, while vgRNA and reovirus infection both induce a similar IRF-dependent gene expression program, gene expression driven by the NF-B family of transcription factors is lower in infected cells. Potent agonists of NF-B such as tumor necrosis factor alpha (TNF-α) and vgRNA failed to induce NF-B-dependent gene expression in infected cells. We demonstrate that NF-B signaling is blocked due to loss of critical members of the inhibitor of kappa B kinase (IKK) complex, NF-B essential modifier (NEMO), and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-B, thereby preventing gene expression. Our study demonstrates that reovirus infection selectively blocks NF-B, likely to counteract its antiviral effects and promote efficient viral replication.

IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-B family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-B is inactive. Further, we demonstrate that NF-B is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-B function. Based on previous evidence that active NF-B limits reovirus infection, we conclude that inactivating NF-B is a viral strategy to produce a cellular environment that is favorable for virus replication.

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3D^pol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.

IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3D^pol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

The nuclear factor kappa B (NF-B) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-B, illustrated by proteasome-dependent degradation of the inhibitory NF-B regulator IB and nuclear translocation and phosphorylation of the NF-B subunit p65. PRV-induced persistent activation of NF-B does not result in expression of negative feedback loop genes, like the gene for IBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-B activation. Hence, PRV infection triggers persistent NF-B activation in an unorthodox way and dramatically modulates the NF-B signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-B signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.

IMPORTANCE The NF-B transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-B, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-B activation shares some mechanistic features with canonical NF-B activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IB kinase (IKK) and even renders infected cells resistant to canonical NF-B activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-B activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-B activation.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.

IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)₃] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.

IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler’s (NeST) showed no difference in vivo and in vitro. Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.

IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.

IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9^H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the A56 virus, the G9^H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9^H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9^H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9^H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9^H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.

IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter’s G9 subcomponent. Although the G9^H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.

IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

The entry/fusion complex (EFC) consists of 11 conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here, we describe serially passaging VACV in nonpermissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection, indicating the occurrence and enrichment of adaptive mutations. Sequencing of genomes of passaged and cloned viruses revealed mutations near the N terminus of the G9 open reading frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His-to-Arg mutation at amino acid 44 or a duplication of amino acids 26 to 39. An adaptive Tyr-to-Cys substitution at amino acid 42 was discovered using error-prone PCR to generate additional mutations. Myristoylation of G9 was unaffected by the near-N-terminal mutations. The roles of the G9 mutations in enhancing plaque size were validated by homologous recombination. The mutants exhibited enhanced entry and spread in A56/K2 cells and induced syncytia at neutral pH in HeLa cells despite the expression of A56/K2. The data suggest that the mutations perturb the interaction of G9 with A56/K2, although some association was still detected in detergent-treated infected cell lysates.

IMPORTANCE The entry of enveloped viruses is achieved by the fusion of viral and cellular membranes, a critical step in infection that determines host range and provides targets for vaccines and therapeutics. Poxviruses encode an exceptionally large number of proteins comprising the entry/fusion complex (EFC), which enables infection of diverse cells. Vaccinia virus (VACV), the prototype member of the poxvirus family, also encodes the fusion regulatory proteins A56 and K2, which are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are nonpermissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae. A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family Siphoviridae.

IMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

The rabbit hemorrhagic disease virus (RHDV), which belongs to the family Caliciviridae and the genus Lagovirus, causes lethal fulminant hepatitis in rabbits. RHDV decreases the activity of antioxidant enzymes regulated by Nrf2 in the liver. Antioxidants are important for the maintenance of cellular integrity and cytoprotection. However, the mechanism underlying the regulation of the Nrf2-antioxidant response element (ARE) signaling pathway by RHDV remains unclear. Using isobaric tags for relative and absolute quantification (iTRAQ) technology, the current study demonstrated that RHDV inhibits the induction of ARE-regulated genes and increases the expression of the p50 subunit of the NF-B transcription factor. We showed that RHDV replication causes a remarkable increase in reactive oxygen species (ROS), which is simultaneously accompanied by a significant decrease in Nrf2. It was found that nuclear translocation of Keap1 plays a key role in the nuclear export of Nrf2, leading to the inhibition of Nrf2 transcriptional activity. The p50 protein partners with Keap1 to form the Keap1-p50/p65 complex, which is involved in the nuclear translocation of Keap1. Moreover, upregulation of Nrf2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed rabbit deaths due to RHDV infection. Considered together, our findings suggest that RHDV inhibits the Nrf2-dependent antioxidant response via nuclear translocation of Keap1-NF-B complex and nuclear export of Nrf2 and provide new insight into the importance of oxidative stress during RHDV infection.

IMPORTANCE Recent studies have reported that rabbit hemorrhagic disease virus (RHDV) infection reduced Nrf2-related antioxidant function. However, the regulatory mechanisms underlying this process remain unclear. The current study showed that the NF-B p50 subunit partners with Keap1 to form the Keap1-NF-B complex, which plays a key role in the inhibition of Nrf2 transcriptional activity. More importantly, upregulated Nrf2 activity delayed the death of RHDV-infected rabbits, strongly indicating the importance of oxidative damage during RHDV infection. These findings may provide novel insights into the pathogenesis of RHDV.

Although pain is a prevalent nonmotor symptom in Parkinson’s disease (PD), it is undertreated, in part because of our limited understanding of the underlying mechanisms. Considering that the basal ganglia are implicated in pain sensation, and that their synaptic outputs are controlled by the subthalamic nucleus (STN), we hypothesized that...

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme...

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g.,...

Aerosol–radiation interaction (ARI) plays a significant role in the accumulation of fine particulate matter (PM2.5) by stabilizing the planetary boundary layer and thus deteriorating air quality during haze events. However, modification of photolysis by aerosol scattering or absorbing solar radiation (aerosol–photolysis interaction or API) alters the atmospheric oxidizing capacity, decreases...

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking...

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire–mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled...

Active matter, both synthetic and biological, demonstrates complex spatiotemporal self-organization and the emergence of collective behavior. A coherent rotational motion, the vortex phase, is of great interest because of its ability to orchestrate well-organized motion of self-propelled particles over large distances. However, its generation without geometrical confinement has been a...

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such “early activation” genes silent have been a mystery. In the course...

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the...

Functioning cells depend on the outward-facing plasma membrane (PM) effectively contacting the endoplasmic reticulum (ER), which serves as a central hub for contacts with mitochondria and other intracellular organelles. The contact sites are critical to intracellular communication because they mediate intermembrane exchange of lipids, ions, and other small molecules that...

Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro. Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.

Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl) kinase, has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A (PP2A), it plays a crucial role in activating one of the most important mitotic kinases, known as cyclin-dependent kinase 1 (CDK1). MASTL has been seen to be upregulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop, but the complete mechanism of its activation is still unclear. Here, we report that AKT phosphorylates MASTL at residue T299, which plays a critical role in its activation. Our results suggest that AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which, in turn, promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation, since AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT plays an important role in the progression of mitosis in mammalian cells and that it does so through the phosphorylation and activation of MASTL.

ABSTRACT

By modulating the structure, diversity, and trophic outputs of microbial communities, phages play crucial roles in many biomes. In oligotrophic polar deserts, the effects of katabatic winds, constrained nutrients, and low water availability are known to limit microbial activity. Although phages may substantially govern trophic interactions in cold deserts, relatively little is known regarding the precise ecological mechanisms. Here, we provide the first evidence of widespread antiphage innate immunity in Antarctic environments using metagenomic sequence data from hypolith communities as model systems. In particular, immunity systems such as DISARM and BREX are shown to be dominant systems in these communities. Additionally, we show a direct correlation between the CRISPR-Cas adaptive immunity and the metavirome of hypolith communities, suggesting the existence of dynamic host-phage interactions. In addition to providing the first exploration of immune systems in cold deserts, our results suggest that phages actively challenge niche communities in Antarctic polar deserts. We provide evidence suggesting that the regulatory role played by phages in this system is an important determinant of bacterial host interactions in this environment.

IMPORTANCE In Antarctic environments, the combination of both abiotic and biotic stressors results in simple trophic levels dominated by microbiomes. Although the past two decades have revealed substantial insights regarding the diversity and structure of microbiomes, we lack mechanistic insights regarding community interactions and how phages may affect these. By providing the first evidence of widespread antiphage innate immunity, we shed light on phage-host dynamics in Antarctic niche communities. Our analyses reveal several antiphage defense systems, including DISARM and BREX, which appear to dominate in cold desert niche communities. In contrast, our analyses revealed that genes which encode antiphage adaptive immunity were underrepresented in these communities, suggesting lower infection frequencies in cold edaphic environments. We propose that by actively challenging niche communities, phages play crucial roles in the diversification of Antarctic communities.

ABSTRACT

Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.

IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

Background:

Direct-to-consumer prescription drug advertising is prevalent and affects patient care. Previous research that examined its effect on the patient-provider relationship predates many changes in the advertising and medical landscape that have occurred in the last decade, such as the rise in online promotion and the push for value-based medicine.

Background:

Despite widespread recognition that adherence to clinical preventive guidelines improves patient outcomes, clinicians struggle to implement guideline changes in a timely manner. Multiple factors influence guideline adoption and effective implementation. However, few studies evaluate their collective and inter-related effects. This qualitative study provides a comprehensive picture of the interplay between multiple factors on uptake of new or changed preventive guidelines.

Background:

Facilitation is an effective approach for helping practices implement sustainable evidence-based practice improvements. Few studies examine the facilitation infrastructure and support needed for large-scale dissemination and implementation initiatives.

Zhi-Jun Zhao, Davina Derous, Abby Gerrard, Jing Wen, Xue Liu, Song Tan, Catherine Hambly, and John R. Speakman

Lactating mice increase food intake 4- to 5-fold, reaching an asymptote in late lactation. A key question is whether this asymptote reflects a physiological constraint, or a maternal investment strategy (a ‘restraint’). We exposed lactating mice to periods of food restriction, hypothesizing that if the limit reflected restraint, they would compensate by breaching the asymptote when refeeding. In contrast, if it was a constraint, they would by definition be unable to increase their intake on refeeding days. Using isotope methods, we found that during food restriction, the females shut down milk production, impacting offspring growth. During refeeding, food intake and milk production rose again, but not significantly above unrestricted controls. These data provide strong evidence that asymptotic intake in lactation reflects a physiological/physical constraint, rather than restraint. Because hypothalamic neuropeptide Y (Npy) was upregulated under both states of restriction, this suggests the constraint is not imposed by limits in the capacity to upregulate hunger signalling (the saturated neural capacity hypothesis). Understanding the genetic basis of the constraint will be a key future goal and will provide us additional information on the nature of the constraining factors on reproductive output, and their potential links to life history strategies.

Christopher T. Richards and Enrico A. Eberhard

Muscle force-length dynamics are governed by intrinsic contractile properties, motor stimulation and mechanical load. Although intrinsic properties are well-characterised, physiologists lack in vitro instrumentation accounting for combined effects of limb inertia, musculoskeletal architecture and contractile dynamics. We introduce in vitro virtual-reality (in vitro-VR) which enables in vitro muscle tissue to drive a musculoskeletal jumping simulation. In hardware, muscle force from a frog plantaris was transmitted to a software model where joint torques, inertia and ground reaction forces were computed to advance the simulation at 1 kHz. To close the loop, simulated muscle strain was returned to update in vitro length. We manipulated 1) stimulation timing and, 2) the virtual muscle's anatomical origin. This influenced interactions among muscular, inertial, gravitational and contact forces dictating limb kinematics and jump performance. We propose that in vitro-VR can be used to illustrate how neuromuscular control and musculoskeletal anatomy influence muscle dynamics and biomechanical performance.

Yi Huang, Jazmin Osorio Mendoza, Catherine Hambly, Baoguo Li, Zengguang Jin, Li Li, Moshen Madizi, Sumei Hu, and John R. Speakman

The heat dissipation limit theory predicts lactating female mice consuming diets with lower specific dynamic action (SDA) should have enhanced lactation performance. Dietary fat has lower SDA than other macronutrients. Here we tested the effects of graded dietary fat levels on lactating Swiss mice. We fed females five diets varying in fat content from 8.3 to 66.6%. Offspring of mothers fed diets of 41.7% fat and above were heavier and fatter at weaning compared to those of 8.3% and 25% fat diets. Mice on dietary fat contents of 41.7% and above had greater metabolizable energy intake at peak lactation (8.3%: 229.4±39.6, 25%: 278.8±25.8, 41.7%: 359.6±51.5, 58.3%: 353.7±43.6, 66.6%: 346±44.7 kJ day^–1), lower daily energy expenditure (8.3%: 128.5±16, 25%: 131.6±8.4, 41.7%: 124.4±10.8, 58.3%: 115.1±10.5, 66.6%: 111.2±11.5 kJ day^–1) and thus delivered more milk energy to their offspring (8.3%: 100.8±27.3, 25%: 147.2±25.1, 41.7%: 225.1±49.6, 58.3%: 238.6±40.1, 66.6%: 234.8±41.1 kJ day^–1). Milk fat content (%) was unrelated to dietary fat content, indicating females on higher fat diets (> 41.7%) produced more rather than richer milk. Mothers consuming diets with 41.7% fat or above enhanced their lactation performance compared to those on 25% or less, probably by diverting dietary fat directly into the milk, thereby avoiding the costs of lipogenesis. At dietary fat contents above 41.7% they were either unable to transfer more dietary fat to the milk, or they chose not to do so, potentially because of a lack of benefit to the offspring that were increasingly fatter as maternal dietary fat increased.

Lakshman Abhilash, Arshad Kalliyil, and Vasu Sheeba

Even though the rhythm in adult emergence and rhythm in locomotor activity are two different rhythmic phenomena that occur at distinct life-stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. In an earlier study, we have shown that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, we were interested in asking if temperature sensitivity of the locomotor activity rhythm has evolved in our populations with divergent timing of adult emergence rhythm, with the goal of understanding the extent of similarity (or lack of it) in circadian organisation between the two rhythms. We found that in response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrain faster than early chronotypes (populations selected for predominant emergence during dawn) to 6-h phase-delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time-cues). Additionally, we found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature sensitive activity/rest rhythms. Our results highlight remarkably similar organisation principles between emergence and activity/rest rhythms.

Thomas J. Lisney, Shaun P. Collin, and Jennifer L. Kelley

Ecological factors such as spatial habitat complexity and diet can explain variation in visual morphology, but few studies have sought to determine whether visual specialisation can occur among populations of the same species. We used a small Australian freshwater fish (the western rainbowfish, Melanotaenia australis) to determine whether populations showed variation in eye size and eye position, and whether this variation could be explained by environmental (light availability, turbidity) and ecological (predation risk, habitat complexity, invertebrate abundance) variables. We investigated three aspects of eye morphology, (1) eye size relative to body size, (2) pupil size relative to eye size, and (3) eye position in the head, for fish collected from 14 sites in a major river catchment in northwest Western Australia. We found significant variation among populations in all three measures of eye morphology, but no effect of sex on eye size or eye position. Variation in eye diameter and eye position was best explained by the level of habitat complexity. Specifically, fish occurring in habitats with low complexity (i.e. open water) tended to have smaller, more dorsally-located eyes, than those occurring in more complex habitats (i.e. vegetation present). The size of the pupil relative to the size of the eye was most influenced by the presence of surrounding rock formations; fish living in gorge habitats had significantly smaller pupils (relative to eye size) than those occupying semi-gorge sites or open habitats. Our findings reveal that different ecological and environmental factors contribute to habitat-specific visual specialisations within a species.

Nadia Vicenzi, Alejandro Laspiur, Paola L. Sassi, Ruben Massarelli, John Krenz, and Nora R. Ibargüengoytia

In ectotherms, temperature exerts a strong influence on the performance of physiological and ecological traits. One approach to understand the impact of rising temperatures on animals and their ability to cope with climate change is to quantify variation in thermal-sensitive traits. Here, we examined the thermal biology, the temperature dependence and the thermal plasticity of bite force (endurance and magnitude) in Diplolaemus leopardinus, an aggressive and territorial lizard, endemic to Mendoza province, Argentina. Our results indicated that this lizard behaves like a moderate thermoregulator which uses the rocks of its environment as the main heat source. Bite endurance was not influenced by head morphometry and body temperature, whereas bite force was influenced by head length and jaw length, and exhibited thermal dependence. Before thermal acclimation treatments, the maximum bite force for D. leopardinus occured at the lowest body temperature and fell sharply with increasing body temperature. After acclimation treatments, lizards acclimated at higher temperatures exhibited greater bite force. Bite force showed phenotypic plasticity, which reveals that leopard iguanas are able to maintain (and even improve) their bite force under a rising-temperature scenario.

Background

Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient.

Many noncoding RNAs are known to play a role in the cell directly linked to their structure. Structure prediction based on the sole sequence is, however, a challenging task. On the other hand, thanks to the low cost of sequencing technologies, a very large number of homologous sequences are becoming available for many RNA families. In the protein community, the idea of exploiting the covariance of mutations within a family to predict the protein structure using the direct-coupling-analysis (DCA) method has emerged in the last decade. The application of DCA to RNA systems has been limited so far. We here perform an assessment of the DCA method on 17 riboswitch families, comparing it with the commonly used mutual information analysis and with state-of-the-art R-scape covariance method. We also compare different flavors of DCA, including mean-field, pseudolikelihood, and a proposed stochastic procedure (Boltzmann learning) for solving exactly the DCA inverse problem. Boltzmann learning outperforms the other methods in predicting contacts observed in high-resolution crystal structures.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'–3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'–3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

BACKGROUND:The threshold of the lower limit of the normal range of lung function has been suggested to be more accurate than the 0.7 fixed ratio (FEV1/FVC < 0.7) for a diagnosis of COPD. We aimed to explore the health status and risk factors of patients overdiagnosed with COPD when using the lower limit of the normal range as a diagnostic reference.METHODS:Subjects with COPD diagnosed by a pulmonologist according to guidelines of the Global Initiative for Chronic Obstructive Lung Disease were recruited from October 2016 to April 2018. Overdiagnosed COPD was defined as FEV1/FVC that meets the criterion of the 0.7 fixed ratio but not the the lower limit of the normal range criterion. Spirometry and questionnaires were performed by eligible subjects.RESULTS:Of the 513 subjects included in the final analysis, 20 (3.9%) were overdiagnosed when using the lower limit of the normal range as the diagnostic reference. The subjects who were overdiagnosed were older, weighed more, had better lung function, lower modified Medical British Research Council scores, and higher St. George's Respiratory Questionnaire and 36-item Short Form Survey scores than the subjects who were correctly diagnosed. Older age, heavier weight, exposure to cooking oil fumes, or a new-built or newly renovated home were associated with an increased risk of overdiagnosis of COPD (age adjusted odds ratio (OR) 1.17, 95% CI 1.09–1.26; weight adjusted OR 1.08, 95% CI 1.03–1.13; exposure to cooking oil fumes adjusted OR 3.00, 95% CI, 1.04–8.68; exposure to new-built or newly renovated home adjusted OR 10.88, 95% CI 1.46–80.87.CONCLUSIONS:The subjects with overdiagnosed COPD had a better health status and lung function than the subjects who were correctly diagnosed. Older age, heavier weight, and exposure to cooking oil fumes or a new-built or newly renovated home were factors associated with the overdiagnosis of COPD. These findings may help reduce overdiagnosis of COPD.