Search Results (39)

Coronavirus non-structural protein 1 (nsp1) is a pathogenic determinant of Betacoronaviruses. Previous studies demonstrated that the nsp1 of various coronaviruses induces host shutoff through a variety of mechanisms; however, there is little information on the function of bovine coronavirus (BCoV) nsp1. We aimed to characterize the host gene expression suppression function of BCoV nsp1. We first confirmed that the expression of BCoV nsp1 in MAC-T cells, a bovine mammary epithelial cell line, suppressed host and reporter gene expression. Subsequently, lysine and phenylalanine at amino acid positions 232 and 233, respectively, were identified as key residues required for this suppressive effect. Expression levels of housekeeping genes are comparable in cells expressing wild-type BCoV nsp1 and a mutant with alanine substitutions at positions 232 and 233 (BCoV nsp1-KF). Wild-type BCoV nsp1 localized to both the cytoplasm and nucleus; however, BCoV nsp1-KF exhibited prominent nuclear accumulation with dot-like structures. Using confocal microscopy and co-sedimentation analysis, we identified an association between wild-type BCoV nsp1, but not BCoV nsp1-KF, and ribosomes, suggesting that ribosome binding is required for BCoV nsp1-mediated suppression of host gene expression. This is the first study of the characterization of host gene expression suppression by BCoV nsp1. Full article

The papers introduced in the Commentary present new insights and review aspects of current knowledge concerning the competition between viruses and their hosts for the cellular translation apparatus. Viruses depend on this apparatus and utilize diverse mechanisms to usurp it for the translation of viral mRNAs and to suppress synthesis of cellular proteins. Virus-induced modification of translation factors, selective abrogation of mRNA binding to ribosomes and degradation of cellular mRNAs all impair elements of the innate immune response, thereby undermining host defenses against infection. Various cellular mechanisms prevent translation of viral mRNAs, by modifying components of the translation apparatus to effect a generalized shut-off of translation or by binding of host proteins to viral mRNAs to induce their degradation or to prevent their engagement with the translation apparatus. Viruses have in turn evolved countermeasures to evade these defenses, for example by encoding proteins that impair the activity of host factors or via alterations in the sequence and structure of viral mRNAs. Such changes enable viral mRNAs to avoid recognition by host factors or to support translation initiation by specialized mechanisms that involve only a subset of the factors that are required by cellular mRNAs. Full article

Alphaviruses can replicate in arthropods and in many vertebrate species including humankind, but only in vertebrate cells do infections with these viruses result in a strong inhibition of host translation and transcription. Translation shutoff by alphaviruses is a multifactorial process that involves both host- and virus-induced mechanisms, and some of them are not completely understood. Alphavirus genomes contain cis-acting elements (RNA structures and dinucleotide composition) and encode protein activities that promote the translational and transcriptional resistance to type I IFN-induced antiviral effectors. Among them, IFIT1, ZAP and PKR have played a relevant role in alphavirus evolution, since they have promoted the emergence of multiple viral evasion mechanisms at the translational level. In this review, we will discuss how the adaptations of alphaviruses to vertebrate hosts likely involved the acquisition of new features in viral mRNAs and proteins to overcome the effect of type I IFN. Full article

Arboviruses are defined by their ability to replicate in both mosquito vectors and mammalian hosts. There is good evidence that arboviruses “prime” their progeny for infection of the next host, such as via differential glycosylation of their outer glycoproteins or packaging of host ribosomal subunits. We and others have previously shown that mosquito-derived viruses more efficiently infect mammalian cells than mammalian-derived viruses. These observations are consistent with arboviruses acquiring host-specific adaptations, and we hypothesized that a virus derived from either the mammalian host or mosquito vector elicits different responses when infecting the mammalian host. Here, we perform an RNA-sequencing analysis of the transcriptional response of Human Embryonic Kidney 293 (HEK-293) cells to infection with either mosquito (Aedes albopictus, C7/10)- or mammalian (Baby Hamster Kidney, BHK-21)-derived Sindbis virus (SINV). We show that the C7/10-derived virus infection leads to a more robust transcriptional response in HEK-293s compared to infection with the BHK-derived virus. Surprisingly, despite more efficient infection, we found an increase in interferon-β (IFN-β) and interferon-stimulated gene (ISG) transcripts in response to the C7/10-derived virus infection versus the BHK-derived virus infection. However, translation of interferon-stimulated genes was lower in HEK-293s infected with the C7/10-derived virus, starkly contrasting with the transcriptional response. This inhibition of ISG translation is reflective of a more rapid overall shut-off of host cell translation following infection with the C7/10-derived virus. Finally, we show that the C7/10-derived virus infection of HEK-293 cells leads to elevated levels of phosphorylated eukaryotic translation elongation factor-2 (eEF2), identifying a potential mechanism leading to the more rapid shut-off of host translation. We postulate that the rapid shut-off of host translation in mammalian cells infected with the mosquito-derived virus acts to counter the IFN-β-stimulated transcriptional response. Full article

Herpes simplex virus 2 (HSV-2) causes most sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective (ICP8-) HSV stimulates immune responses in animals without producing progeny virus, making it potentially useful as a safe form of a live vaccine against HSV. We previously demonstrated that mice generate a stronger response to ICP8- virus encoding B7-2 costimulation molecules than to the parental replication-defective virus. We have also demonstrated enhanced immunogenicity of an ICP8-, virion host shutoff (vhs)- virus which can no longer destabilize viral and host mRNAs. Here, we constructed a triple mutant, ICP8-vhs-B7-2+ strain, and compared it to both double mutant viruses. Immunization of mice with a single dose of ICP8-B7-2+ or ICP8-vhs-B7-2+ virus decreased challenge virus replication in the vaginal mucosa, genital disease, and mortality more effectively than immunization with the ICP8-vhs- virus. Immunization with ICP8-B7-2+ or ICP8-vhs-B7-2+ virus also effectively suppressed subsequent HSV-2 infection of the nervous system compared to immunization with the ICP8-vhs- virus. ICP8-B7-2+ and ICP8-vhs-B7-2+ strains induced more IFN gamma-producing CD8 T cells and memory CD8 T cells than did ICP8-vhs- virus, potentially explaining the enhanced protective effects. Thus, B7 costimulation molecules expressed from a replication-defective vaccine can enhance vaccine efficacy, even in an immunocompetent host. Full article

Spraying systems to protect crops against pests are still necessary to maintain food production at the rates demanded by the current population. However, today, it is crucial to use precision agriculture to reduce the negative effects of pesticides and other agrochemicals such as fungicides. In particular, pressure fluctuations related to transient states when using pulse-width-modulated nozzles (PMW) have been reported to decrease the accuracy of preset flow rates in air-assisted orchard sprayers. The objective of this paper is to analyze the vibrations induced in the spraying system of a vineyard blast sprayer controlled by pulse-width-modulated nozzles, considering the instantaneous duty cycle (DC) as the control variable. An air-assisted vineyard sprayer was modified to host 24 solenoid shutoff valves with hollow disc–cone nozzles. A triaxial accelerometer was mounted to track the effect of duty cycle (20%, 30%, 50%, and 70%). In addition to accelerations, high-speed images were recorded, and the pressure according to time and the flow were estimated. The hydraulic system of the sprayer, when controlled in real time by the PWM solenoids, created pulsating impacts at the nozzle level with the same frequency of 10 Hz of the PMW system. The impact effect was significantly higher for low duty cycles under 40% DC. In addition, to demonstrate the inaccuracy of opening and closing the valves at a precisely specified time, this study also confirmed the divergence between the theoretical duty cycles commanded by the sprayer’s control unit and the actual ones measured in real time. The results of the analysis showed the difficulty of opening and closing the valves with precision to obtain accurate duty cycles in the practical implementation of smart sprayers and the importance of understanding the vibration effects of pulses in arrangements of multiple PWM nozzles working simultaneously. Full article

African swine fever is a viral disease of swine caused by the African swine fever virus (ASFV). Currently, ASFV is spreading over the Eurasian continent and threatening global pig husbandry. One viral strategy to undermine an efficient host cell response is to establish a global shutoff of host protein synthesis. This shutoff has been observed in ASFV-infected cultured cells using two-dimensional electrophoresis combined with metabolic radioactive labeling. However, it remained unclear if this shutoff was selective for certain host proteins. Here, we characterized ASFV-induced shutoff in porcine macrophages by measurement of relative protein synthesis rates using a mass spectrometric approach based on stable isotope labeling with amino acids in cell culture (SILAC). The impact of ASFV infection on the synthesis of >2000 individual host proteins showed a high degree of variability, ranging from complete shutoff to a strong induction of proteins that are absent from naïve cells. GO-term enrichment analysis revealed that the most effective shutoff was observed for proteins related to RNA metabolism, while typical representatives of the innate immune system were strongly induced after infection. This experimental setup is suitable to quantify a virion-induced host shutoff (vhs) after infection with different viruses. Full article

During reactivation from latency, gammaherpesviruses radically restructure their host cell to produce virion particles. To achieve this and thwart cellular defenses, they induce rapid degradation of cytoplasmic mRNAs, suppressing host gene expression. In this article, we review mechanisms of shutoff by Epstein–Barr virus (EBV) and other gammaherpesviruses. In EBV, canonical host shutoff is accomplished through the action of the versatile BGLF5 nuclease expressed during lytic reactivation. We explore how BGLF5 induces mRNA degradation, the mechanisms by which specificity is achieved, and the consequences for host gene expression. We also consider non-canonical mechanisms of EBV-induced host shutoff. Finally, we summarize the limitations and barriers to accurate measurements of the EBV host shutoff phenomenon. Full article

Human immunodeficiency virus 1 (HIV-1) viral protease (PR) is one of the most studied viral enzymes and a crucial antiviral target. Despite its well-characterized role in virion maturation, an increasing body of research is starting to focus on its ability to cleave host cell proteins. Such findings are apparently in contrast with the dogma of HIV-1 PR activity being restricted to the interior of nascent virions and suggest catalytic activity within the host cell environment. Given the limited amount of PR present in the virion at the time of infection, such events mainly occur during late viral gene expression, mediated by newly synthesized Gag-Pol polyprotein precursors, rather than before proviral integration. HIV-1 PR mainly targets proteins involved in three different processes: those involved in translation, those controlling cell survival, and restriction factors responsible for innate/intrinsic antiviral responses. Indeed, by cleaving host cell translation initiation factors, HIV-1 PR can impair cap-dependent translation, thus promoting IRES-mediated translation of late viral transcripts and viral production. By targeting several apoptotic factors, it modulates cell survival, thus promoting immune evasion and viral dissemination. Additionally, HIV-1 PR counteracts restriction factors incorporated in the virion that would otherwise interfere with nascent virus vitality. Thus, HIV-1 PR appears to modulate host cell function at different times and locations during its life cycle, thereby ensuring efficient viral persistency and propagation. However, we are far from having a complete picture of PR-mediated host cell modulation, which is emerging as a field that needs further investigation. Full article

Viruses are obligatorily intracellular pathogens. To generate progeny virus particles, influenza A viruses (IAVs) have to divert the cellular machinery to ensure sufficient translation of viral mRNAs. To this end, several strategies have been exploited by IAVs, such as host gene shutoff, suppression of host innate immune responses, and selective translation of viral mRNAs. Various IAV proteins are responsible for host gene shutoff, e.g., NS1, PA-X, and RdRp, through inhibition of cellular gene transcription, suppression of cellular RNA processing, degradation of cellular RNAs, and blockage of cellular mRNA export from the nucleus. Host shutoff should suppress the innate immune responses and also increase the translation of viral mRNAs indirectly due to the reduced competition from cellular mRNAs for cellular translational machinery. However, many other mechanisms are also responsible for the suppression of innate immune responses by IAV, such as prevention of the detection of the viral RNAs by the RLRs, inhibition of the activities of proteins involved in signaling events of interferon production, and inhibition of the activities of interferon-stimulated genes, mainly through viral NS1, PB1-F2, and PA-X proteins. IAV mRNAs may be selectively translated in favor of cellular mRNAs through interacting with viral and/or cellular proteins, such as NS1, PABPI, and/or IFIT2, in the 5′-UTR of viral mRNAs. This review briefly summarizes the strategies utilized by IAVs to ensure sufficient translation of viral mRNAs focusing on recent developments. Full article

Chikungunya virus (CHIKV) causes outbreaks of rash, arthritis, and fever associated with neurologic complications, where astrocytes are preferentially infected. A determinant of virulence is the macrodomain (MD) of nonstructural protein 3 (nsP3), which binds and removes ADP-ribose (ADPr) from ADP-ribosylated substrates and regulates stress-granule disruption. We compared the replication of CHIKV 181/25 (WT) and MD mutants with decreased ADPr binding and hydrolase (G32S) or increased ADPr binding and decreased hydrolase (Y114A) activities in C8-D1A astrocytic cells and NSC-34 neuronal cells. WT CHIKV replication was initiated more rapidly with earlier nsP synthesis in C8-D1A than in NSC-34 cells. G32S established infection, amplified replication complexes, and induced host-protein synthesis shut-off less efficiently than WT and produced less infectious virus, while Y114A replication was close to WT. However, G32S mutation effects on structural protein synthesis were cell-type-dependent. In NSC-34 cells, E2 synthesis was decreased compared to WT, while in C8-D1A cells synthesis was increased. Excess E2 produced by G32S-infected C8-D1A cells was assembled into virus particles that were less infectious than those from WT or Y114A-infected cells. Because nsP3 recruits ADP-ribosylated RNA-binding proteins in stress granules away from translation-initiation factors into nsP3 granules where the MD hydrolase can remove ADPr, we postulate that suboptimal translation-factor release decreased structural protein synthesis in NSC-34 cells while failure to de-ADP-ribosylate regulatory RNA-binding proteins increased synthesis in C8-D1A cells. Full article

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis. Full article

Infectious hematopoietic necrosis virus (IHNV) is a pathogen that causes high rates of mortality in salmonid fishes. Therefore, an RNA-seq-based transcriptome analysis was performed in the head kidney of rainbow trout infected with a highly virulent IHNV strain to understand the pathogenesis of and defense strategies for IHNV infection in rainbow trout. The results showed that the numbers of DEGs were 618, 2626, and 774 (control vs. IHNV) on days 1, 3, and 5, respectively. Furthermore, the enrichment analysis of gene ontology (GO) annotations to classify DEGs showed that GO terms considerably associated with DEGs were gluconeogenesis, inflammatory response, and cell adhesion in the Biological Process (BP) category, apical plasma membrane, extracellular matrix (ECM) in the Cellular Component category, and transporter activity, integrin binding, and protein homodimerization activity in the Molecular Function category, on days 1, 3, and 5, respectively. Notably, GO terms in the BP category, including the negative regulation of type I interferon production and positive regulation of interleukin-1β secretion, were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, complement and coagulation cascades were commonly identified at all time points. Importantly, the widely recognized GO terms and KEGG pathways extensively linked to DEGs were related to energy metabolism on day 1, the immune response on day 3, and cell proliferation on day 5. Furthermore, protein–protein interaction networks and centrality analysis showed that the metabolism and signaling transduction pathways were majorly upregulated. Conclusively, the virulent IHNV infection drives pathogenesis by activating the metabolic energy pathway for energy use for viral replication, facilitating necrosis through autophagy, and causing a shutoff response of the host immune system through the downregulation of type I IFN at the initial stage of infection. Full article

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32, and both phages encode a protein similar to bacterial RNA polymerase promoter specificity σ subunit. Here, we investigated the temporal pattern of 7-11 gene expression during infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays, we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11 σ subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus, GTAAtg-(16)-aCTA, and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzymes formed by the phi32_gp36 σ factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by the RNA polymerase holoenzyme containing the host primary σ⁷⁰ factor. Unlike the case of phiEco32, no shut-off of σ⁷⁰-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme. Full article

The Madin–Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing. Full article