The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or ...pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
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•PAC-MAN is a CRISPR-based strategy for RNA-guided viral RNA inhibition and degradation•Cas13d PAC-MAC is effective at targeting and cleaving SARS-CoV-2 sequences•Cas13d PAC-MAC can reduce H1N1 IAV load in respiratory epithelial cells•A group of six crRNAs can target more than 90% of all coronaviruses
A CRISPR-based strategy is developed to target conserved sequences across coronaviruses and other pathogenic viruses.
Sex hormones, such as estrogen and testosterone, are steroid compounds with well-characterized effects on the coordination and development of vertebrate reproductive systems. Since their discovery, ...however, it has become clear that these "sex hormones" also regulate/influence a broad range of biological functions. In this review, we will summarize some current findings on how estrogens interact with and regulate inflammation and immunity. Specifically, we will focus on describing the mechanisms by which estrogens alter immune pathway activation, the impact of these changes during infection and the development of long-term immunity, and how different types of estrogens and their respective concentrations mediate these outcomes.
Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the ...host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) ...expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A “reader” protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.
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•m6A sites on influenza A virus (IAV) mRNAs and vRNAs were mapped•High levels of m6A modification increase IAV RNA expression•IAV mutants lacking m6A sites on the HA segment are attenuated in culture•These same IAV HA m6A mutants show reduced pathogenicity in mice
Influenza A virus (IAV) transcripts bear numerous epitranscriptomic m6A modifications. Courtney et al. map these modifications on both the IAV mRNA and vRNA strands and demonstrate that m6A increases viral RNA expression in cis. Moreover, IAV mutants lacking HA sites on the viral HA segment show reduced pathogenicity in vivo.
The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight ...viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The molecular basis for the diversity across influenza strains is poorly understood. To gain insight into this question, we mutagenized the viral genome and sequenced recoverable viruses. Only two ...small regions in the genome were enriched for insertions, the hemagglutinin head and the immune-modulatory nonstructural protein 1. These proteins play a major role in host adaptation, and thus need to be able to evolve rapidly. We propose a model in which certain influenza A virus proteins (or protein domains) exist as highly plastic scaffolds, which will readily accept mutations yet retain their functionality. This model implies that the ability to rapidly acquire mutations is an inherent aspect of influenza HA and nonstructural protein 1 proteins; further, this may explain why rapid antigenic drift and a broad host range is observed with influenza A virus and not with some other RNA viruses.
...these cells appear to mediate the temporary window of antigen-independent immunity against unrelated viruses after IAV infection has resolved, which has been described in human populations 9. ...There are numerous other potential roles for cells derived from unappreciated fates. Since these cells are not being cleared from the body, if they are able to retain and harbor even low amounts of virally derived protein long term, they could be significant contributors to a process like B cell affinity maturation. Is the idea of a “typical” cell fate an artificial concept derived from work in immortalized cell culture systems? Because of their rapid growth rates (and indefinite proliferation), immortalized cell culture lines (HeLa, Vero, A549, etc.) have long been the standard medium with which to not only propagate virus but also study the nature of viral infection. ...there is much work to be done to lay the groundwork for even a basic understanding of the cellular populations that survive cytopathic viral infection.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular ...pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.
Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have ...been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses.
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•CRISPR activation used in a genome-wide screen for viral restriction factors•B4GALNT2 was the major hit in the screen, and activation restricted IAV infection•B4GALNT2 modifies glycans containing α2,3-linked sialic acids•Glycan modification by B4GALNT2 restricted infection by all tested avian IAV strains
Heaton et al. apply CRISPR SAM genome-wide screening technology to find restriction factors for avian and human strains of influenza A virus. The major hit, B4GALNT2, modified a glycan containing the influenza A virus receptor and restricted infection with all avian strains tested.
Highlights ► Viruses physically remodel the cell by manipulating lipid signaling and synthesis. ► Viruses manipulate cellular lipid metabolism to facilitate replication and assembly. ► Lipid ...synthesis is emerging as a target of intrinsic and innate antiviral immunity.
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