DeePhafier: a phage lifestyle classifier using a multilayer self-attention neural network combining protein information.

Bacteriophages are the viruses that infect bacterial cells. They are the most diverse biological entities on earth and play important roles in microbiome. According to the phage lifestyle, phages can be divided into the virulent phages and the temperate phages. Classifying virulent and temperate phages is crucial for further understanding of the phage-host interactions. Although there are several methods designed for phage lifestyle classification, they merely either consider sequence features or gene features, leading to low accuracy. A new computational method, DeePhafier, is proposed to improve classification performance on phage lifestyle. Built by several multilayer self-attention neural networks, a global self-attention neural network, and being combined by protein features of the Position Specific Scoring Matrix matrix, DeePhafier improves the classification accuracy and outperforms two benchmark methods. The accuracy of DeePhafier on five-fold cross-validation is as high as 87.54% for sequences with length >2000bp.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

The battle between host antiviral innate immunity and immune evasion by cytomegalovirus.

Cytomegalovirus (CMV) has successfully established a long-lasting latent infection in humans due to its ability to counteract the host antiviral innate immune response. During coevolution with the host, the virus has evolved various evasion techniques to evade the host's innate immune surveillance. At present, there is still no vaccine available for the prevention and treatment of CMV infection, and the interaction between CMV infection and host antiviral innate immunity is still not well understood. However, ongoing studies will offer new insights into how to treat and prevent CMV infection and its related diseases. Here, we update recent studies on how CMV evades antiviral innate immunity, with a focus on how CMV proteins target and disrupt critical adaptors of antiviral innate immune signaling pathways. This review also discusses some classic intrinsic cellular defences that are crucial to the fight against viral invasion. A comprehensive review of the evasion mechanisms of antiviral innate immunity by CMV will help investigators identify new therapeutic targets and develop vaccines against CMV infection.

Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Discovery and characterization of a novel LysinB from F2 sub-cluster mycobacteriophage RitSun.

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum.

Mouse cytomegalovirus lacking sgg1 shows reduced import into the salivary glands.

Cytomegaloviruses (CMVs) transmit via chronic shedding from the salivary glands. How this relates to the broad cell tropism they exhibit in vitro is unclear. Human CMV (HCMV) infection presents only after salivary gland infection is established. Murine CMV (MCMV) is therefore useful to analyse early infection events. It reaches the salivary glands via infected myeloid cells. Three adjacent spliced genes designated as m131/129 (MCK-2), sgg1 and sgg1.1, positional homologues of the HCMV UL128/130/131 tropism determinants, are implicated. We show that a sgg1 null mutant is defective in infected myeloid cell entry into the salivary glands, a phenotype distinct from MCMV lacking MCK-2. These data point to a complex, multi-step process of salivary gland colonization.

Multi-omics characterization of the monkeypox virus infection.

Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-ß pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.

Structures of African swine fever virus topoisomerase complex and their implications.

African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.

Insights into the structure of RNPs from segmented negative-sense RNA viruses.

The genome of segmented negative-sense single-stranded RNA viruses, such as influenza virus and bunyaviruses, is coated by viral nucleoproteins (NPs), forming a ribonucleoprotein (RNP). In this issue of Structure, Dick et al.1 expand our knowledge on the RNPs of these viruses by solving the structures of Thogoto virus NP and RNP.

Oxysterole-binding protein targeted by SARS-CoV-2 viral proteins regulates coronavirus replication.

Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Phage lysins for intestinal microbiome modulation: current challenges and enabling techniques.

The importance of the microbiota in the intestinal tract for human health has been increasingly recognized. In this perspective, microbiome modulation, a targeted alteration of the microbial composition, has gained interest. Phage lysins, peptidoglycan-degrading enzymes encoded by bacteriophages, are a promising new class of antibiotics currently under clinical development for treating bacterial infections. Due to their high specificity, lysins are considered microbiome-friendly. This review explores the opportunities and challenges of using lysins as microbiome modulators. First, the high specificity of endolysins, which can be further modulated using protein engineering or targeted delivery methods, is discussed. Next, obstacles and possible solutions to assess the microbiome-friendliness of lysins are considered. Finally, lysin delivery to the intestinal tract is discussed, including possible delivery methods such as particle-based and probiotic vehicles. Mapping the hurdles to developing lysins as microbiome modulators and identifying possible ways to overcome these hurdles can help in their development. In this way, the application of these innovative antimicrobial agents can be expanded, thereby taking full advantage of their characteristics.

Inverted Sandwich-Type e-LCR Aided by Lambda Exonuclease-RecJf Combination Enables Ultrasensitive Detection of Low-Frequency EGFR-L858R Mutation in NSCLC.

Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.

An enterococcal phage protein inhibits type IV restriction enzymes involved in antiphage defense.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

Mining and multifaceted applications of phage lysin for combatting Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a prevalent foodborne pathogen found in both water and seafood, poses substantial risks to public health. The conventional countermeasure, antibiotics, has exacerbated the issue of antibiotic resistance, increasing the difficulty of controlling this bacterium. Phage lysins, as naturally occurring active proteins, offer a safe and reliable strategy to mitigate the impact of V. parahaemolyticus on public health. However, there is currently a research gap concerning bacteriophage lysins specific to Vibrio species. To address this, our study innovatively and systematically evaluates 37 phage lysins sourced from the NCBI database, revealing a diverse array of conserved domains and notable variations in similarity among Vibrio phage lysins. Three lysins, including Lyz_V_pgrp, Lyz_V_prgp60, and Lyz_V_zlis, were successfully expressed and purified. Optimal enzymatic activity was observed at 45℃, 800 mM NaCl, and pH 8-10, with significant enhancements noted in the presence of 1 mM membrane permeabilizers such as EDTA or organic acids. These lysins demonstrated effective inhibition against 63 V. parahaemolyticus isolates from clinical, food, and environmental sources, including the reversal of partial resistance, synergistic interactions with antibiotics, and disruption of biofilms. Flow cytometry analyses revealed that the combination of Lyz_V_pgp60 and gentamicin markedly increased bacterial killing rates. Notably, Lyz_V_pgrp, Lyz_V_pgp60, and Lyz_V_zlis exhibited highly efficient biofilm hydrolysis, clearing over 90 % of preformed V. parahaemolyticus biofilms within 48 h. Moreover, these lysins significantly reduced bacterial loads in various food samples and environmental sources, with reductions averaging between 1.06 and 1.29 Log CFU/cm2 on surfaces such as stainless-steel and bamboo cutting boards and approximately 0.87 CFU/mL in lake water and sediment samples. These findings underscore the exceptional efficacy and versatile application potential of phage lysins, offering a promising avenue for controlling V. parahaemolyticus contamination in both food and environmental contexts.

The PB2 I714S mutation influenced mammalian adaptation of the H3N2 canine influenza virus by interfering with nuclear import efficiency and RNP complex assembly.

Avian influenza viruses (AIVs) are the origin of multiple mammal influenza viruses. The genetic determinants of AIVs adapted to humans have been widely elucidated, however, the molecular mechanism of cross-species transmission and adaptation of AIVs to canines are still poorly understood. In this study, two H3N2 influenza viruses isolated from a live poultry market (A/environment/Guangxi/13431/2018, GX13431) and a swab sample from a canine (A/canine/Guangdong/0601/2019, GD0601) were used to investigate the possible molecular basis that determined H3N2 AIV adapting to canine. We found that GD0601 exhibited more robust polymerase activity in cells and higher pathogenicity in mice compared with its evolution ancestor H3N2 AIV GX13431. A series of reassortments of the ribonucleoprotein (RNP) complex showed that the PB2 subunit was the crucial factor that conferred high polymerase activity of GD0601, and the substitution of I714S in the PB2 subunit of GD0601 attenuated the replication and pathogenicity in mammal cells and the mouse model. Mechanistically, the reverse mutation of I714S in the PB2 polymerase subunit which was identified in AIV GX13431 reduced the nuclear import efficiency of PB2 protein and interfered with the interactions of PB2-PA/NP that affected the assembly of the viral RNP complex. Our study reveals amino acid mutation at the position of 714 in the nuclear localization signal (NLS) area in PB2 plays an important role in overcoming the barrier from poultry to mammals of the H3N2 canine influenza virus and provides clues for further study of mammalian adaptation mechanism of AIVs.

Structures of Mature and Urea-Treated Empty Bacteriophage T5: Insights into Siphophage Infection and DNA Ejection.

T5 is a siphophage that has been extensively studied by structural and biochemical methods. However, the complete in situ structures of T5 before and after DNA ejection remain unknown. In this study, we used cryo-electron microscopy (cryo-EM) to determine the structures of mature T5 (a laboratory-adapted, fiberless T5 mutant) and urea-treated empty T5 (lacking the tip complex) at near-atomic resolutions. Atomic models of the head, connector complex, tail tube, and tail tip were built for mature T5, and atomic models of the connector complex, comprising the portal protein pb7, adaptor protein p144, and tail terminator protein p142, were built for urea-treated empty T5. Our findings revealed that the aforementioned proteins did not undergo global conformational changes before and after DNA ejection, indicating that these structural features were conserved among most myophages and siphophages. The present study elucidates the underlying mechanisms of siphophage infection and DNA ejection.

DepoScope: Accurate phage depolymerase annotation and domain delineation using large language models.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.