Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Control of lysogeny and antiphage defense by a prophage-encoded kinase-phosphatase module.

The filamentous 'Pf' bacteriophages of Pseudomonas aeruginosa play roles in biofilm formation and virulence, but mechanisms governing Pf prophage activation in biofilms are unclear. Here, we identify a prophage regulatory module, KKP (kinase-kinase-phosphatase), that controls virion production of co-resident Pf prophages and mediates host defense against diverse lytic phages. KKP consists of Ser/Thr kinases PfkA and PfkB, and phosphatase PfpC. The kinases have multiple host targets, one of which is MvaU, a host nucleoid-binding protein and known prophage-silencing factor. Characterization of KKP deletion and overexpression strains with transcriptional, protein-level and prophage-based approaches indicates that shifts in the balance between kinase and phosphatase activities regulate phage production by controlling MvaU phosphorylation. In addition, KKP acts as a tripartite toxin-antitoxin system that provides defense against some lytic phages. A conserved lytic phage replication protein inhibits the KKP phosphatase PfpC, stimulating toxic kinase activity and blocking lytic phage production. Thus, KKP represents a phosphorylation-based mechanism for prophage regulation and antiphage defense. The conservation of KKP gene clusters in >1000 diverse temperate prophages suggests that integrated control of temperate and lytic phage infection by KKP-like regulatory modules may play a widespread role in shaping host cell physiology.

Potential Transcriptional Enhancers in Coronaviruses: From Infectious Bronchitis Virus to SARS-CoV-2.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Identification of the Promoter Antisense Transcript Enhancing the Transcription of the Equine Herpesvirus-1 Immediate-Early Gene.

Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion, and encephalomyelitis in horses. The EHV-1 immediate-early (IE) protein, essential for viral replication, is transactivated by the binding of a multiprotein complex including the open reading frame 12 (ORF12) and some host factors to the IE promoter region. Promoter-associated non-coding RNAs (pancRNAs), which are transcribed from bidirectional promoters, regulate the transcription of neighboring genes in mammals and pathogens. In this study, we identified a novel pancRNA transcribed from across the areas of the 5'-untranslated region and a promoter of EHV-1 IE and named it IE pancRNA. IE pancRNA and mRNA were simultaneously expressed in EHV-1-infected RN33B-A68B2M cells. This pancRNA was also transcribed in RK13 and E. Derm cells, which are highly susceptible to EHV-1 infection. Furthermore, IE pancRNA upregulated IE gene expression in the presence of ORF12, and stable expression of IE pancRNA increased the number of EHV-1-infected RN33B-A68B2M cells. These results suggest that IE pancRNAs facilitate EHV-1 proliferation by promoting IE gene expression.

Transcription of Endogenous Retroviruses: Broad and Precise Mechanisms of Control.

Endogenous retroviruses (ERVs) are the remnants of retroviral germline infections and are highly abundant in the genomes of vertebrates. At one time considered to be nothing more than inert 'junk' within genomes, ERVs have been tolerated within host genomes over vast timescales, and their study continues to reveal complex co-evolutionary histories within their respective host species. For example, multiple instances have been characterized of ERVs having been 'borrowed' for normal physiology, from single copies to ones involved in various regulatory networks such as innate immunity and during early development. Within the cell, the accessibility of ERVs is normally tightly controlled by epigenetic mechanisms such as DNA methylation or histone modifications. However, these silencing mechanisms of ERVs are reversible, and epigenetic alterations to the chromatin landscape can thus lead to their aberrant expression, as is observed in abnormal cellular environments such as in tumors. In this review, we focus on ERV transcriptional control and draw parallels and distinctions concerning the loss of regulation in disease, as well as their precise regulation in early development.

Duck plague virus-encoded microRNA dev-miR-D28-3p inhibits viral replication via targeting UL27.

Herpesviruses-encoded microRNAs (miRNAs) have been discovered to be essential regulators in viral life cycle, participating in viral replication, latent or lytic infection, and immunological escape. However, the roles of miRNAs encoded by duck plague virus (DPV) are still unknown. Dev-miR-D28-3p is a miRNA uniquely encoded by DPV CHv strain. The aim of this study was to explore the effect of dev-miR-D28-3p on DPV replication and explore the potential mechanisms involved. Our findings demonstrated that transfection of dev-miR-D28-3p mimic into duck embryo fibroblasts (DEFs) effectively suppressed viral copies, viral titers and viral protein expressions during DPV infection, while the results above were reversed after transfection with dev-miR-D28-3p inhibitor. Subsequently, we further discovered that dev-miR-D28-3p specifically bound to DPV-encoded UL27 and inhibited its expression, suggesting that UL27 was the target gene of dev-miR-D28-3p. Finally, we investigated the role of UL27 in DPV replication and found the overexpression of UL27 increased viral copies, viral titers, and viral protein expressions; whereas the opposite results appear when knockdown of UL27. Our findings illustrated a novel mechanism that DPV regulated itself replication via dev-miR-D28-3p, paving the way for exploring the role of DPV-encoded miRNAs.

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway.

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

Structural basis of phage transcriptional regulation.

Phages are the most prevalent and diverse entities in the biosphere and represent the simplest systems that are capable of self-replication. Many fundamental concepts of transcriptional regulation were revealed through phage studies. The replication of phages within bacteria entails the hijacking of the host transcription machinery. Typically, this is accomplished through proteins and RNAs encoded by the phage genome that bind to the host RNA polymerase and modify its characteristics. Understanding these processes offers valuable insights into the mechanisms of bacterial transcription itself. Historically, X-ray crystallography has been the major tool for elucidating the structural basis of phage transcriptional regulation. In recent years, the application of cryoelectron microscopy has not only allowed the exploration of protein-protein and protein-nucleic acid interactions at near-atomic resolution but also captured transient intermediate states, further expanding our mechanistic understanding of phage transcriptional regulation.

Impact of the interaction between herpes simplex virus 1 ICP22 and FACT on viral gene expression and pathogenesis.

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.

AcMNPV-miR-2 affects Autographa californica nucleopolyhedrovirus infection by regulating the expression of ac28 and several other viral early genes.

Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration. IMPORTANCE: Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.

Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposi's sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

ORC1 enhances repressive epigenetic modifications on HIV-1 LTR to promote HIV-1 latency.

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs. IMPORTANCE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Human cytomegalovirus microRNAs: strategies for immune evasion and viral latency.

Viruses use various strategies and mechanisms to deal with cells and proteins of the immune system that form a barrier against infection. One of these mechanisms is the encoding and production of viral microRNAs (miRNAs), whose function is to regulate the gene expression of the host cell and the virus, thus creating a suitable environment for survival and spreading viral infection. miRNAs are short, single-stranded, non-coding RNA molecules that can regulate the expression of host and viral proteins, and due to their non-immunogenic nature, they are not eliminated by the cells of the immune system. More than half of the viral miRNAs are encoded and produced by Orthoherpesviridae family members. Human cytomegalovirus (HCMV) produces miRNAs that mediate various processes in infected cells to contribute to HCMV pathogenicity, including immune escape, viral latency, and cell apoptosis. Here, we discuss which cellular and viral proteins or cellular pathways and processes these mysterious molecules target to evade immunity and support viral latency in infected cells. We also discuss current evidence that their function of bypassing the host's innate and adaptive immune system is essential for the survival and multiplication of the virus and the spread of HCMV infection.

Exploring the anti-EBV potential of suberoylanilide hydroxamic acid: Induction of apoptosis in infected cells through suppressing BART gene expression and inducing lytic infection.

Epstein-Barr virus (EBV) is linked to lymphoma and epithelioma but lacks drugs specifically targeting EBV-positive tumors. BamHI A Rightward Transcript (BART) miRNAs are expressed in all EBV-positive tumors, suppressing both lytic infection and host cell apoptosis. We identified suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase enzymes, as an agent that suppresses BART promoter activity and transcription of BART miRNAs. SAHA treatment demonstrated a more pronounced inhibition of cell proliferation in EBV-positive cells compared to EBV-negative cells, affecting both p53 wild-type and mutant gastric epithelial cells. SAHA treatment enhanced lytic infection in wild-type EBV-infected cells, while also enhancing cell death in BZLF1-deficient EBV-infected cells. It reduced BART gene expression by 85% and increased the expression of proapoptotic factors targeted by BART miRNAs. These findings suggest that SAHA not only induces lytic infection but also leads to cell death by suppressing BART miRNA transcription and promoting the apoptotic program.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

The Distal Promoter of the B438L Gene of African Swine Fever Virus Is Responsible for the Transcription of the Alternatively Spliced B169L.

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L. Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA (B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92-169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV.

Analyzing the Impact of the Highest Expressed Epstein-Barr Virus-Encoded microRNAs on the Host Cell Transcriptome.

The Epstein-Barr virus (EBV) has a very high prevalence (>90% in adults), establishes a lifelong latency after primary infection, and exerts an oncogenic potential. This dsDNA virus encodes for various molecules, including microRNAs (miRs), which can be detected in the latent and lytic phases with different expression levels and affect, among others, immune evasion and malignant transformation. In this study, the different EBV miRs are quantified in EBV-positive lymphomas, and the impact on the host cell transcriptome of the most abundant EBV miRs will be analyzed using comparative RNA sequencing analyses. The EBV miRs ebv-miR-BART1, -BART4, -BART17, and -BHRF1-1 were most highly expressed, and their selective overexpression in EBV-negative human cells resulted in a large number of statistically significantly down- and up-regulated host cell genes. Functional analyses showed that these dysregulated target genes are involved in important cellular processes, including growth factor pathways such as WNT, EGF, FGF, and PDGF, as well as cellular processes such as apoptosis regulation and inflammation. Individual differences were observed between these four analyzed EBV miRs. In particular, ebv-miR-BHRF1-1 appears to be more important for malignant transformation and immune evasion than the other EBV miRs.

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.