Oligoadenylate-Synthetase-Family Protein OASL Inhibits Activity of the DNA Sensor cGAS during DNA Virus Infection to Limit Interferon Production.

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.

"Transfer" of power: The intersection of DNA virus infection and tRNA biology.

Transfer RNAs (tRNAs) are at the heart of the molecular biology central dogma, functioning to decode messenger RNAs into proteins. As obligate intracellular parasites, viruses depend on the host translation machinery, including host tRNAs. Thus, the ability of a virus to fine-tune tRNA expression elicits the power to impact the outcome of infection. DNA viruses commonly upregulate the output of RNA polymerase III (Pol III)-dependent transcripts, including tRNAs. Decades after these initial discoveries we know very little about how mature tRNA pools change during viral infection, as tRNA sequencing methodology has only recently reached proficiency. Here, we review perturbation of tRNA biogenesis by DNA virus infection, including an emerging player called tRNA-derived fragments (tRFs). We discuss how tRNA dysregulation shifts the power landscape between the host and virus, highlighting the potential for tRNA-based antivirals as a future therapeutic.

RNF144A promotes antiviral responses by modulating STING ubiquitination.

Stimulator of interferon (IFN) genes (STING, also named MITA, ERIS, MPYS, or TMEM173) plays an essential role in DNA virus- or cytosolic DNA-triggered innate immune responses. Here, we demonstrate that the RING-in-between RING (RBR) E3 ubiquitin ligase family member RING-finger protein (RNF) 144A interacts with STING and promotes its K6-linked ubiquitination at K236, thereby enhancing STING translocation from the ER to the Golgi and downstream signaling pathways. The K236R mutant of STING displays reduced activity in promoting innate immune signal transduction. Overexpression of RNF144A upregulates HSV-1- or cytosolic DNA-induced immune responses, while knockdown of RNF144A expression has the opposite effect. In addition, Rnf144a-deficient cells exhibit impaired DNA virus- or cytosolic DNA-triggered signaling, and RNF144A protects mice from DNA virus infection. In contrast, RNF144A does not affect RNA virus- or cytosolic RNA-triggered innate immune responses. Taken together, our findings identify a new positive regulator of DNA virus- or cytosolic DNA-triggered signaling pathways and a critical ubiquitination site important for fully functional STING during antiviral responses.

Circular RNAs in Immune Response and Viral Infection.

Circular RNAs (circRNAs) are a diverse class of RNAs with varying sizes, cellular abundance, and biological functions. Investigations from the past decade have revealed that circRNAs are ubiquitously found in eukaryotes and have defined the different biological roles of circRNAs to illuminate this previously unrecognized class of molecules. In the context of the immune system, immune responses and immune-related diseases alter circRNA expression. More recently, several oncogenic double-stranded DNA viruses have been found to encode circRNAs. In this review, we summarize the current understanding of circRNAs and their emerging functions in immune regulation and autoimmune disorders, and discuss the identification and potential roles of viral circRNAs during infections. Finally, we present promising areas for future investigations in the nascent field of circRNAs.

Regulation of Virus Replication by BK Polyomavirus Small T Antigen.

Polyomavirus small T antigen (tAg) plays important roles in regulating viral replication, the innate immune response, apoptosis, and transformation for SV40, Merkel cell polyomavirus (MCPyV), murine polyomavirus (MuPyV), and JC polyomavirus (JCPyV). However, the function of BK polyomavirus (BKPyV) tAg has been much less studied. Here, we constructed mutant viruses that do not express tAg, and we showed that, in contrast with other polyomaviruses, BKPyV tAg inhibits large T antigen (TAg) gene expression and viral DNA replication. However, this occurs only in an archetype viral background. We also observed that the transduction of cells with a lentivirus-expressing BKPyV tAg kills the cells. We further discovered that BKPyV tAg interacts not only with PP2A A and C subunits, as has been demonstrated for other polyomavirus tAg proteins, but also with PP2A B''' subunit members. Knocking down either of two B''' subunits, namely STRN or STRN3, mimics the phenotype of the tAg mutant virus. However, a virus containing a point mutation in the PP2A binding domain of tAg only partially affected virus TAg expression and DNA replication. These results indicate that BKPyV tAg downregulates viral gene expression and DNA replication and that this occurs in part through interactions with PP2A. IMPORTANCE BK polyomavirus is a virus that establishes a lifelong infection of the majority of people. The infection usually does not cause any clinical symptoms, but, in transplant recipients whose immune systems have been suppressed, unchecked virus replication can cause severe disease. In this study, we show that a viral protein called small T antigen is one of the ways that the virus can persist without high levels of replication. Understanding which factors control viral replication enhances our knowledge of the virus life cycle and could lead to potential interventions for these patients.

RNA recognition by PKR during DNA virus infection.

Protein kinase R (PKR) is a double-stranded RNA (dsRNA) binding protein that plays a crucial role in innate immunity during viral infection and can restrict both DNA and RNA viruses. The potency of its antiviral function is further reflected by the large number of viral-encoded PKR antagonists. However, much about the regulation of dsRNA accumulation and PKR activation during viral infection remains unknown. Since DNA viruses do not have an RNA genome or RNA replication intermediates like RNA viruses do, PKR-mediated dsRNA detection in the context of DNA virus infection is particularly intriguing. Here, we review the current state of knowledge regarding the regulation of PKR activation and its antagonism during infection with DNA viruses.

Dinucleotide biases in the genomes of prokaryotic and eukaryotic dsDNA viruses and their hosts.

The genomes of cellular organisms display CpG and TpA dinucleotide composition biases. Such biases have been poorly investigated in dsDNA viruses. Here, we show that in dsDNA virus, bacterial, and eukaryotic genomes, the representation of TpA and CpG dinucleotides is strongly dependent on genomic G + C content. Thus, the classical observed/expected ratios do not fully capture dinucleotide biases across genomes. Because a larger portion of the variance in TpA frequency was explained by G + C content, we explored which additional factors drive the distribution of CpG dinucleotides. Using the residuals of the linear regressions as a measure of dinucleotide abundance and ancestral state reconstruction across eukaryotic and prokaryotic virus trees, we identified an important role for phylogeny in driving CpG representation. Nonetheless, phylogenetic ANOVA analyses showed that few host associations also account for significant variations. Among eukaryotic viruses, most significant differences were observed between arthropod-infecting viruses and viruses that infect vertebrates or unicellular organisms. However, an effect of viral DNA methylation status (either driven by the host or by viral-encoded methyltransferases) is also likely. Among prokaryotic viruses, cyanobacteria-infecting phages resulted to be significantly CpG-depleted, whereas phages that infect bacteria in the genera Burkolderia and Staphylococcus were CpG-rich. Comparison with bacterial genomes indicated that this effect is largely driven by the general tendency for phages to resemble the host's genomic CpG content. Notably, such tendency is stronger for temperate than for lytic phages. Our data shed light into the processes that shape virus genome composition and inform manipulation strategies for biotechnological applications.

MYBBP1A is required for efficient replication and gene expression of herpes simplex virus 1.

More than 100 different herpes simplex virus 1 (HSV-1) genes belong to three major classes, and their expression is coordinately regulated and sequentially ordered in a cascade. This complex HSV-1 gene expression is thought to be regulated by various viral and host cellular proteins. A host cellular protein, Myb-binding protein 1A (MYBBP1A), has been reported to be associated with HSV-1 viral genomes in conjunction with viral and cellular proteins critical for DNA replication, repair, and transcription within infected cells. However, the role(s) of MYBBP1A in HSV-1 infections remains unclear. In this study, we examined the effects of MYBBP1A depletion on HSV-1 infection and found that MYBBP1A depletion significantly reduced HSV-1 replication, as well as the accumulation of several viral proteins. These results suggest that MYBBP1A is an important host cellular factor that contributes to HSV-1 replication, plausibly by promoting viral gene expression.

Assembly and Evolution of Poxviruses.

Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).

Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.

Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target a different site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing a fluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16 pM, which is a decrease of the LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.

Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

[Poxvirus-encoded DNA replication proteins: potential targets for antivirals]. / Le complexe de réplication des poxvirus : cible potentielle de molécules antivirales.

In the spring of 2022, an epidemic due to human monkeypox virus (MPXV) of unprecedented magnitude spread across all continents. Although this event was surprising in its suddenness, the resurgence of a virus from the Poxviridae family is not surprising in a world population that has been largely naïve to these viruses since the eradication of the smallpox virus in 1980 and the concomitant cessation of vaccination. Since then, a vaccine and two antiviral compounds have been developed to combat a possible return of smallpox. However, the use of these treatments during the 2022 MPXV epidemic showed certain limitations, indicating the importance of continuing to develop the therapeutic arsenal against these viruses. For several decades, efforts to understand the molecular mechanisms involved in the synthesis of the DNA genome of these viruses have been ongoing. Although many questions remain unanswered up to now, the three-dimensional structures of essential proteins, and in particular of the DNA polymerase holoenzyme in complex with DNA, make it possible to consider the development of a model for poxvirus DNA replication. In addition, these structures are valuable tools for the development of new antivirals targeting viral genome synthesis. This review will first present the molecules approved for the treatment of poxvirus infections, followed by a review of our knowledge of the replication machinery of these viruses. Finally, we will describe how these proteins could be the target of new antiviral compounds.

Giant Viruses Encode Actin-Related Proteins.

The emergence of the eukaryotic cytoskeleton is a critical yet puzzling step of eukaryogenesis. Actin and actin-related proteins (ARPs) are ubiquitous components of this cytoskeleton. The gene repertoire of the Last Eukaryotic Common Ancestor (LECA) would have therefore harbored both actin and various ARPs. Here, we report the presence and expression of actin-related genes in viral genomes (viractins) of some Imitervirales, a viral order encompassing the giant Mimiviridae. Phylogenetic analyses suggest an early recruitment of an actin-related gene by viruses from ancient protoeukaryotic hosts before the emergence of modern eukaryotes, possibly followed by a back transfer that gave rise to eukaryotic actins. This supports a coevolutionary scenario between pre-LECA lineages and their viruses, which could have contributed to the emergence of the modern eukaryotic cytoskeleton.

Ataxia Telangiectasia-Mutated Is Activated but Not Required for Productive Autographa californica Multiple Nucleopolyhedrovirus Infection.

Multiplication of the invertebrate DNA baculoviruses activates the host DNA damage response (DDR), which promotes virus DNA replication. DDR signaling is initiated by the host insect's phosphatidylinositol-3 kinase-related kinases (PIKKs), including ataxia telangiectasia-mutated kinase (ATM). Like other PIKKs, ATM phosphorylates an array of host DDR proteins at serine/threonine glutamine (S/TQ) motifs, the result of which leads to cell cycle arrest, DNA repair, or apoptosis. To define the role of host PIKKs in baculovirus replication, we compared replication levels of the baculovirus prototype species Autographa californica multiple nucleopolyhedrovirus in permissive Spodoptera frugiperda (SF21) cells with and without ATM function. Caffeine, which inhibits multiple DDR kinases, and the ATM-specific inhibitors KU-55933 and KU-60019 each prevented phosphorylation of Spodoptera histone H2AX (SfH2AX), a recognized indicator of ATM activity. However, only caffeine reduced autographa californica multiple nucleopolyhedrovirus (AcMNPV)-induced bulk phosphorylation of S/TQ protein motifs. Furthermore, only caffeine, not KU-55933 or KU-60019, reduced AcMNPV yields, suggesting a limited role for ATM. To investigate further, we identified and edited the Spodoptera ATM gene (sfatm). Consistent with ATM's known functions, CRISPR/Cas9-mediated knockout of sfatm eliminated DNA damage-induced phosphorylation of DDR marker SfH2AX in SF21 cells. However, loss of sfatm failed to affect the levels of AcMNPV multiplication. These findings suggested that in the absence of the kinase SfATM, another caffeine-sensitive host DDR kinase promotes S/TQ phosphorylation and baculovirus multiplication. Thus, baculoviruses activate and utilize the host insect DDR in an ATM-independent manner. IMPORTANCE The DDR, while necessary for the maintenance and fidelity of the host genome, represents an important cellular response to viral infection. The prolific DNA baculoviruses activate and manipulate the invertebrate DDR by using mechanisms that positively impact virus multiplication, including virus DNA replication. As the key DDR initiator kinase, ATM was suspected to play a critical role in this host response. However, we show here that baculovirus AcMNPV activates an ATM-independent DDR. By identifying the insect host ATM ortholog (Spodoptera frugiperda SfATM) and evaluating genetic knockouts, we show that SfATM is dispensable for AcMNPV activation of the DDR and for virus replication. Thus, another PIKK, possibly the closely related kinase ATR (ATM- and Rad3-related kinase), is responsible for efficient baculovirus multiplication. These findings better define the host pathways used by invertebrates to engage viral pathogens, including DNA viruses.

Establishment of a system to quantify wild-type herpes simplex virus-induced cell-cell fusion reveals a role of N-glycosylation of HSV-1 envelope glycoprotein B in cell-cell fusion.

Wild-type herpes simplex virus (HSV) strains infrequently mediate cell-cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell-cell fusion induced by wild-type HSV strains. The established system clarified that the HSV-1 envelope glycoprotein B and its N-glycosylation at asparagine at position 141 were required for efficient cell-cell fusion. This study provides a link between cell-cell fusion induced by wild-type HSV-1 and viral pathogenesis in vivo.

N6-methyladenosine modification-a key player in viral infection.

N6-methyladenosine (m6A) modification is a dynamic, reversible process and is the most prevalent internal modification of RNA. This modification is regulated by three protein groups: methyltransferases ("writers"), demethylases ("erasers"), and m6A-binding proteins ("readers"). m6A modification and related enzymes could represent an optimal strategy to deepen the epigenetic mechanism. Numerous reports have suggested that aberrant modifications of m6A lead to aberrant expression of important viral genes. Here, we review the role of m6A modifications in viral replication and virus-host interactions. In particular, we focus on DNA and RNA viruses associated with human diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV)-1, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV). These findings will contribute to the understanding of the mechanisms of virus-host interactions and the design of future therapeutic targets for treatment of tumors associated with viral infections.

Oral shedding of herpesviruses and clinical outcomes in hematopoietic stem cell transplant patients.

OBJECTIVES: To characterize the oral shedding of herpes viruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and investigate its relationship with clinical outcomes. MATERIALS AND METHODS: Polymerase chain reaction and enzymatic digestion were performed to identify the oral shedding of the members of the Herpesviridae family in 31 patients. The samples were collected from the oral cavity at five timestamps. RESULTS: The presence of each herpesvirus in the oral cavity was observed in 3.2%, 12.9%, 19.3%, 32.2%, 54.8% and 93.5% patients for human herpesvirus (HHV)-6A, herpes simplex virus-1, HHV-6B, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and HHV-7, respectively. Oral shedding of herpes virus was not uncommon after alloHSCT. There was a statistically significant association between the EBV and CMV oral shedding at C1 and the cumulative incidence of acute graft-versus-host disease (aGVHD). The results suggested that the presence of HSV-1 at C2 was related to a relapse. The HHV-7 oral shedding at C2 suggests a possible link between relapse, progression-free survival and overall survival of the patients. CONCLUSIONS: Patients who developed aGVHD showed higher CMV and EBV shedding in the oral cavity at aplasia, suggesting modifications to the pattern of immune cell response and inflammatory microenvironment.

KAT5 acetylates cGAS to promote innate immune response to DNA virus.

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA-triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus-induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus.

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry.

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.

A plant DNA virus replicates in the salivary glands of its insect vector via recruitment of host DNA synthesis machinery.

Whereas most of the arthropod-borne animal viruses replicate in their vectors, this is less common for plant viruses. So far, only some plant RNA viruses have been demonstrated to replicate in insect vectors and plant hosts. How plant viruses evolved to replicate in the animal kingdom remains largely unknown. Geminiviruses comprise a large family of plant-infecting, single-stranded DNA viruses that cause serious crop losses worldwide. Here, we report evidence and insight into the replication of the geminivirus tomato yellow leaf curl virus (TYLCV) in the whitefly (Bemisia tabaci) vector and that replication is mainly in the salivary glands. We found that TYLCV induces DNA synthesis machinery, proliferating cell nuclear antigen (PCNA) and DNA polymerase δ (Polδ), to establish a replication-competent environment in whiteflies. TYLCV replication-associated protein (Rep) interacts with whitefly PCNA, which recruits DNA Polδ for virus replication. In contrast, another geminivirus, papaya leaf curl China virus (PaLCuCNV), does not replicate in the whitefly vector. PaLCuCNV does not induce DNA-synthesis machinery, and the Rep does not interact with whitefly PCNA. Our findings reveal important mechanisms by which a plant DNA virus replicates across the kingdom barrier in an insect and may help to explain the global spread of this devastating pathogen.