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Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199 , demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1 , a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza. Here, Li et al. perform a genome-wide CRISPR screen to identify host dependency factors for influenza A virus infection and show that the host mRNA cap methyltransferase CMTR1 is important for viral cap snatching and that it affects expression of antiviral genes.

Transcriptome-wide mapping of N1-methyladenosine (m 1 A) at single-nucleotide resolution reveals m 1 A to be scarce in cytoplasmic mRNA, to inhibit translation, and to be highly dynamic at a single site in a mitochondrial mRNA. The basis of m1A modification N 1 -methyladenosine (m 1 A) modification has been detected on mRNA, but validation of the internal mRNA sites at which it occurs and the functional consequences of it have not been well defined. Schraga Schwartz and colleagues now address these limitations using a method that enables single-nucleotide resolution of such sites in the transcriptome. They show that the level of modification is much lower than reported previously and varies during development and by tissue type. The authors identify a structural motif associated with the modification and define the enzymatic machinery responsible for the methylation. They find that m 1 A modification is associated with translational repression, consistent with its tight regulation. Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N 1 -methyladenosine (m 1 A), which disrupts Watson–Crick base pairing, at internal sites of mRNAs 1 , 2 . These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m 1 A at single-nucleotide resolution. Within the cytosol, m 1 A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m 1 A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m 1 A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m 1 A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m 1 A levels was adopted as a potential means of post-transcriptional regulation.

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression. IMPORTANCE Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression. Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression.

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate. Most people around the world unknowingly carry the human cytomegalovirus, as this virus can become dormant after infection and hide in small numbers of blood stem cells (which give rise to blood and immune cells). Dormant viruses still make their host cells read their genetic information and create viral proteins – a process known as gene expression – but they do not use them to quickly multiply. However, it is possible for the cytomegalovirus to reawaken at a later stage and start replicating again, which can be fatal for people with weakened immune systems. It is therefore important to understand exactly how the virus can stay dormant, and how it reactivates. Only certain infected cells allow dormant viruses to later reactivate; in others, it never starts to multiply again. Techniques that can monitor individual cells are therefore needed to understand how the host cells and the viruses interact during dormant infection and reactivation. To investigate this, Shnayder et al. infected blood stem cells in the laboratory and used a method known as single-cell RNA analysis, which highlights all the genes (including viral genes) that are expressed in a cell. This showed that in certain cells, the virus dampens the cell defenses, leading to a higher rate of viral gene expression and, in turn, easier reactivation. Further experiments showed that the blood stem cells that expressed the viral genes were marked to become a type of immune cells known as monocytes. In turn, these infected monocytes were shown to be less able to defend the body against infection, suggesting that latent human cytomegalovirus suppresses the body’s innate immune response. The reactivation of human cytomegalovirus is a dangerous issue for patients who have just received an organ or blood stem cells transplant. The study by Shnayder et al. indicates that treatments that boost innate immunity may help to prevent the virus from reawakening, but more work is needed to test this theory.

Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.

Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5' end, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.

N 6 -methyladenosine (m 6 A) is the most common mRNA modification. Recent studies have revealed that depletion of m 6 A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m 6 A ‘writer’ METTL3 or ‘reader’ YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB , the gene encoding the main cytokine that drives the type I interferon response, was m 6 A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m 6 A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m 6 A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation. RNAs can be dynamically modified by N 6 -methylation of adenosine (m 6 A), which leads to their destabilization. Stern-Ginossar and colleagues demonstrate a role for m 6 A modification of host transcripts encoding type I interferons during viral infection.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error (‘cxgenes’). The correct word is ‘genes’. The error has been corrected in the HTML and PDF version of the article.

Background Aspergillus fumigatus (Af) is one of the most ubiquitous fungi and its infection potency is suggested to be strongly controlled by the host genetic background. The aim of this study was to search for candidate genes associated with host susceptibility to Aspergillus fumigatus (Af) using an RNAseq approach in CC lines and hepatic gene expression. Methods We studied 31 male mice from 25 CC lines at 8 weeks old; the mice were infected with Af. Liver tissues were extracted from these mice 5 days post‐infection, and next‐generation RNA‐sequencing (RNAseq) was performed. The GENE‐E analysis platform was used to generate a clustered heat map matrix. Results Significant variation in body weight changes between CC lines was observed. Hepatic gene expression revealed 12 top prioritized candidate genes differentially expressed in resistant versus susceptible mice based on body weight changes. Interestingly, three candidate genes are located within genomic intervals of the previously mapped quantitative trait loci (QTL), including Gm16270 and Stox1 on chromosome 10 and Gm11033 on chromosome 8. Conclusions Our findings emphasize the CC mouse model's power in fine mapping the genetic components underlying susceptibility towards Af. As a next step, eQTL analysis will be performed for our RNA‐Seq data. Suggested candidate genes from our study will be further assessed with a human cohort with aspergillosis. A graphical representation of the experimental design and outcome.

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.