Suppressing Host Gene Expression Research Articles

Characterization of nsp1 Binding to the Viral RNA Leader Sequence of Severe Acute Respiratory Syndrome Coronavirus.

Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host mRNAs and triggering their decay. Surprisingly, viral RNA, which resembles host mRNAs containing a 5'-cap and a 3'-poly(A) tail, escapes significant translation inhibition and RNA decay, aiding viral propagation. Current literature proposes that, in SCOV2, nsp1 binds the viral RNA leader sequence, and the interaction may serve to distinguish viral RNA from host mRNA. However, a direct binding between SCOV1 nsp1 and the corresponding RNA leader sequence has not been established yet. Here, we show that SCOV1 nsp1 binds to the SCOV1 RNA leader sequence but forms multiple complexes at a high concentration of nsp1. These complexes are marginally different from complexes formed with SCOV2 nsp1. Finally, mutations of the RNA stem-loop did not completely abolish RNA binding by nsp1, suggesting that an RNA secondary structure is more important for binding than the sequence itself. Understanding the nature of binding of nsp1 to viral RNA will allow us to understand how this viral protein selectively suppresses host gene expression.

An Evolutionarily Conserved Strategy for Ribosome Binding and Host Translation Inhibition by β-coronavirus Non-structural Protein 1

An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Non-structural protein 1 (Nsp1), which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from β-Coronaviruses (β-CoV) inhibits translation through ribosome binding. The C-terminal domain (CTD) of all β-CoV Nsp1s confers high-affinity ribosome binding despite low sequence conservation. Modeling of interactions of four Nsp1s with the ribosome identified the few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the β-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1′s N-terminal “effector” domain. Finally, we show that a cis-acting viral RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aid future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2 and related human-pathogenic β-CoVs. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein.

EBV Reactivation from Latency Is a Degrading Experience for the Host.

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.

Nonstructural Protein 1 of SARS Coronavirus Interacts with Stress Granule Protein G3BP1 and Accumulates in the Stress Granule

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) orchestrates a multi‐pronged mechanism to suppress host gene expression, also known as the host shutoff. The C‐terminus of nsp1 competes for the mRNA binding site in the 40S ribosome, preventing host translation from host mRNAs. Additionally, nsp1 triggers selective cleavage of host mRNAs while keeping the viral RNA intact. Nsp1 of SARS‐CoV and SARS‐CoV‐2 share 84% sequence identity, prompting us to believe they share a similar mechanism of host shutoff. So far identification of nsp1‐interacting partners has isolated stable complexes of nsp1 with the 40S and 60S ribosomal proteins. However, these complexes do not reveal any transient interaction nsp1 might have with other host proteins to suppress cellular function. Using proximity‐dependent biotinylation to capture the transient interactions of nsp1 with other cellular proteins, we found multiple subunits of the nuclear pre‐mRNA processing complex and cellular stress granule‐associated proteins. Here, we show that nsp1 interacts with the majority of these pre‐mRNA processing and stress granule‐associated proteins through its C‐terminal domain, the region responsible for binding the 40S ribosome. In addition, nsp1 co‐immunoprecipitates with stress granule‐associated protein G3BP1 and eIF4G. Upon induction of stress, these proteins accumulate in nonmembranous organelles called stress granules that are known to store stalled translation complexes. Using both immunofluorescence and immunoblot against stress granule‐associated proteins we show that nsp1 disrupts the maturation of stress granules.

The Role of SARS‐CoV Nonstructural Protein 1 in Cytoplasmic Stress Granule Formation

Severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) encoded nonstructural protein 1 (nsp1) is known to suppress host gene expression, known as host shutoff. Nsp1 interacts with host proteins that facilitate nsp1 to block host protein synthesis by cellular protein translation factors. Nsp1 also triggers the degradation of host messenger RNA (mRNA) while keeping the viral RNA intact. During viral infection, stalled host mRNAs accumulate in non‐membranous vesicles, known as stress granules (SGs). This allows mRNAs to be protected until stress ceases. Recently, our lab observed that nsp1 interacts with proteins of, and accumulates in, the SGs as well. We found SGs are smaller in size in the presence of nsp1. We aimed to determine the role of nsp1 within these granules. Given the role of nsp1 in degrading mRNAs, we suspected that this viral protein may modify the composition of SGs to prevent it from protecting host mRNAs. To identify any compositional changes by nsp1, we successfully adapted and optimized SG isolation methods in nsp1‐expressing human embryonic kidney (HEK) cells. We then further isolated the RNA following standard protocols for RNA‐seq analysis. Using immunoblot, we analyzed the proteins present in SGs isolated from cells in the presence and absence of nsp1. We were able to further isolate and identify these proteins using immunoblot and found that SG core protein, Ras‐GTPase activating protein (SH3 domain)‐binding protein 1 or G3BP1, decreases overtime in the presence of nsp1. Since nsp1 is responsible for targeting mRNA for degradation we are currently identifying mRNAs that selectively accumulate in SGs in the presence of nsp1.

Proximity-dependent biotinylation detects associations between SARS coronavirus nonstructural protein 1 and stress granule–associated proteins

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)–associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain–binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite–induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy

BackgroundOncolytic virotherapy (OV) is an immunotherapy that incorporates viral cancer cell lysis with engagement of the recruited immune response against cancer cells. Pediatric solid tumors are challenging targets because they...

Retrotransposons as pathogenicity factors of the plant pathogenic fungus Botrytis cinerea

BackgroundRetrotransposons are genetic elements inducing mutations in all domains of life. Despite their detrimental effect, retrotransposons can become temporarily active during epigenetic reprogramming and cellular stress response, which may accelerate host genome evolution. In fungal pathogens, a positive role has been attributed to retrotransposons when shaping genome architecture and expression of genes encoding pathogenicity factors; thus, retrotransposons are known to influence pathogenicity.ResultsWe uncover a hitherto unknown role of fungal retrotransposons as being pathogenicity factors, themselves. The aggressive fungal plant pathogen, Botrytis cinerea, is known to deliver some long-terminal repeat (LTR) deriving regulatory trans-species small RNAs (BcsRNAs) into plant cells to suppress host gene expression for infection. We find that naturally occurring, less aggressive B. cinerea strains possess considerably lower copy numbers of LTR retrotransposons and had lost retrotransposon BcsRNA production. Using a transgenic proof-of-concept approach, we reconstitute retrotransposon expression in a BcsRNA-lacking B. cinerea strain, which results in enhanced aggressiveness in a retrotransposon and BcsRNA expression-dependent manner. Moreover, retrotransposon expression in B. cinerea leads to suppression of plant defence-related genes during infection.ConclusionsWe propose that retrotransposons are pathogenicity factors that manipulate host plant gene expression by encoding trans-species BcsRNAs. Taken together, the novelty that retrotransposons are pathogenicity factors will have a broad impact on studies of host-microbe interactions and pathology.

Proteomic Study of Nonstructural Protein 1 of SARS‐CoV to Identify Its Role in Host Shutoff

Severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to suppress host gene expression, also known as the host shutoff. Current literature suggests that the C‐terminus of nsp1 competes for the mRNA binding site in the 40S ribosome, preventing host translation from host mRNAs. However, several mutations on surface residues of nsp1 are reported to either attenuate or augment its host shutoff function suggesting a role of these surface amino acids in dampening host gene expression. Additionally, nsp1 of SARS‐CoV and SARS‐CoV‐2 share 84% sequence identity, prompting us to believe they share a similar mechanism of host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we isolated host proteins proximal to nsp1 in human epithelial kidney (HEK) cells using the proximity‐dependent biotinylation experiment by expressing nsp1 fused to BIOID2. BioID2 is a promiscuous bacterial biotin ligase protein that attaches biotin to free lysine residues of nearby proteins. Biotin treatment of these cells incorporates biotin tag on proteins proximal to nsp1. Using streptavidin magnetic beads, we isolated these proteins and identified them using mass spectroscopy. Finally, several factors were verified by western blot.By comparing protein complexes, we identified a group of proteins including ribosomal proteins that are likely involved in host shutoff. In addition to the ribosomal proteins, factors implicated in the stress granule formation and RNA processing are identified and verified. These proteins are common in both SARS‐CoV and SARS‐CoV‐2. Currently, we are using similar methods to verify the binding of these proteins to nsp1 mutations in the N‐terminus and C‐terminus.

Mechanisms of Coronavirus Nsp1-Mediated Control of Host and Viral Gene Expression.

Many viruses disrupt host gene expression by degrading host mRNAs and/or manipulating translation activities to create a cellular environment favorable for viral replication. Often, virus-induced suppression of host gene expression, including those involved in antiviral responses, contributes to viral pathogenicity. Accordingly, clarifying the mechanisms of virus-induced disruption of host gene expression is important for understanding virus–host cell interactions and virus pathogenesis. Three highly pathogenic human coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, have emerged in the past two decades. All of them encode nonstructural protein 1 (nsp1) in their genomes. Nsp1 of SARS-CoV and MERS-CoV exhibit common biological functions for inducing endonucleolytic cleavage of host mRNAs and inhibition of host translation, while viral mRNAs evade the nsp1-induced mRNA cleavage. SARS-CoV nsp1 is a major pathogenic determinant for this virus, supporting the notion that a viral protein that suppresses host gene expression can be a virulence factor, and further suggesting the possibility that SARS-CoV-2 nsp1, which has high amino acid identity with SARS-CoV nsp1, may serve as a major virulence factor. This review summarizes the gene expression suppression functions of nsp1 of CoVs, with a primary focus on SARS-CoV nsp1 and MERS-CoV nsp1.

The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner.

The influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection.IMPORTANCE The PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

Helicobacter pylori-Induced Rev-erbα Fosters Gastric Bacteria Colonization by Impairing Host Innate and Adaptive Defense.

Background & AimsRev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown.MethodsRev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα–/– and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ–/– and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays.ResultsRev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and β-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c–Ly6G–CD11b+CD68– myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response.ConclusionsOverall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori.

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.

Lysine 164 is critical for SARS-CoV-2 Nsp1 inhibition of host gene expression.

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

Rhinovirus 2A is the key protease responsible for instigating the early block to gene expression in infected cells.

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

The Influenza A Virus Endoribonuclease PA-X Usurps Host mRNA Processing Machinery to Limit Host Gene Expression

SummaryMany viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.

The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines.

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-β and IFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

Identification of novel amino acid residues of influenza virus PA-X that are important for PA-X shutoff activity by using yeast

The influenza A virus protein PA-X comprises an N-terminal PA region and a C-terminal PA-X-specific region. PA-X suppresses host gene expression, termed shutoff, via mRNA cleavage. Although the endonuclease active site in the N-terminal PA region of PA-X and basic amino acids in the C-terminal PA-X-specific region are known to be important for PA-X shutoff activity, other amino acids may also play a role. Here, we used yeast to identify novel amino acids of PA-X that are important for PA-X shutoff activity. Unlike wild-type PA-X, most PA-X mutants predominantly localized in the cytoplasm, indicating that these mutations decreased the shutoff activity of PA-X by affecting PA-X translocation to the nucleus. Mapping of the identified amino acids onto the N-terminal structure of PA revealed that some of them likely contribute to the formation of the endonuclease active site of PA.

RNA-seq-based digital gene expression analysis reveals modification of host defense responses by rice stripe virus during disease symptom development in Arabidopsis.

BackgroundVirus infection induces and suppresses host gene expression on a global level. Rice stripe virus (RSV) is the type species of the genus Tenuivirus and infects rice and Arabidopsis plants. Microarray-based and next generation sequencing-based transcriptomic approaches have been used to study rice-RSV interactions. However, our knowledge of the response of Arabidopsis plants to RSV infection is limited, and it requires further investigation to determine the similarities (or differences) in virus-host interactions between monocot and dicot hosts infected with RSV.MethodsWe characterized transcriptome changes in Arabidopsis thaliana infected with rice stripe virus (RSV) with RNA-seq based digital gene expression (DGE) analysis. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 14 and 21 days post-infection (dpi) during different stages of symptom development.ResultsWe identified 624 differentially expressed genes (DEGs) in Arabidopsis influenced by RSV at 14 dpi and 21 dpi, among which at 14 dpi, 255 transcripts were induced, and 38 were repressed; at 21 dpi, 146 were induced, and 237 were repressed. Functional annotation indicated that these DEGs were related to multiple biological functions, including defense response, secondary metabolism, protein amino acid phosphorylation and response to abiotic stress.ConclusionsImportantly, the transcription of genes related to host defense systems was activated by RSV infection at an early stage of symptom development (14 dpi), whereas over the infection period (21 dpi), the host defense response systems were suppressed. A total of 52 genes were continuously differentially expressed between the two time points, indicating that the majority of DEGs were transient and unique to a particular time point during symptom development. The DEGs, particularly the defense response genes, identified in this study are candidates suitable for further functional analysis during the RSV-Arabidopsis interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0663-7) contains supplementary material, which is available to authorized users.