Characterization of bacteriophage Henu4_2 lytic for Escherichia coli and its therapeutic efficacy in infection models.

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.

Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22

Utilidad del refractómetro como indicador de la mal posición de una sonda enteral

Soybeans agroindustrial residues as Staphylococcus epidermidis and S. aureus biofilm inhibitors

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from −20 °C to 50 °C) and pH values (pH 3–pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5α Protein, a CCR5 shRNA, and a TAR Decoy

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV.IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

Acinetobacter baumannii is a leading cause of biofilm-associated infections, particularly catheter-related bloodstream infections (CRBSIs) that are mostly recalcitrant to antimicrobial therapy. One approach to reducing the burden of CRBSIs is inhibiting biofilm formation on catheters. Owing to their prodigious microbial diversity, bacterial endophytes might be a valuable source of biosurfactants, which are known for their great capacity to disperse microbial biofilms. With this in mind, our study aimed to screen bacterial endophytes from plants growing on the banks of the River Nile for the production of powerful biosurfactants capable of reducing the ability of A. baumannii to form biofilms on central venous catheters (CVCs). This was tested on multidrug- and extensive drug-resistant (M/XDR) clinical isolates of A. baumannii that belong to high-risk global clones and on a standard strain of A. baumannii ATCC 19606. The drop collapse and oil dispersion assays were employed in screening the cell-free supernatants (CFS) of all endophytes for biosurfactant activity. Of the 44 bacterial endophytes recovered from 10 plants, the CFS of Bacillus amyloliquefaciens Cp24, isolated from Cyperus papyrus, showed the highest biosurfactant activity. The crude biosurfactant extract of Cp24 showed potent antibacterial activity with minimum inhibitory concentrations (MICs) ranging from 0.78 to 1.56 mg/ml. It also showed significant antibiofilm activity (p-value<0.01). Sub-MICs of the extract could reduce biofilm formation by up to 89.59%, while up to 87.3% of the preformed biofilms were eradicated by the MIC. A significant reduction in biofilm formation on CVCs impregnated with sub-MIC of the extract was demonstrated by CV assay and further confirmed by scanning electron microscopy. This was associated with three log10 reductions in adhered bacteria in the viable count assay. GC-MS analysis of the crude biosurfactant extract revealed the presence of several compounds, such as saturated, unsaturated, and epoxy fatty acids, cyclopeptides, and 3-Benzyl-hexahydro-pyrrolo [1, 2-a] pyrazine-1,4-dione, potentially implicated in the potent biosurfactant and antibiofilm activities. In the present study, we report the isolation of a B. amyloliquefaciens endophyte from the plant C. papyrus that produces a biosurfactant with potent antibiofilm activity against MDR/XDR global clones of A. baumannii. The impregnation of CVCs with the biosurfactant was demonstrated to reduce biofilms and, hence, proposed as a potential strategy for reducing CRBSIs.

Open AccessCCS ChemistryCOMMUNICATION1 Jan 2022Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry Siwen Liu†, Lin Zhu†, Guangshan Xie†, Bobo Wing-Yee Mok, Zhu Yang, Shaofeng Deng, Siu-Ying Lau, Pin Chen, Pui Wang, Honglin Chen and Zongwei Cai Siwen Liu† State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Lin Zhu† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 HKBU Shenzhen Institute of Research and Continuing Education, Shenzhen 518000 , Guangshan Xie† State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Bobo Wing-Yee Mok State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Zhu Yang State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 , Shaofeng Deng State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Siu-Ying Lau State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pin Chen State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Pui Wang State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 , Honglin Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077 and Zongwei Cai *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong SAR 999077 Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai 519087 https://doi.org/10.31635/ccschem.021.202000603 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmission electron microscope (TEM) imaging, we analyzed early phosphorylation dynamics within the first 15 min during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry. Based on alterations in the phosphorylation events, we found that kinase activities such as protein kinase C (PKC), interleukin-1 receptor-associated kinase 4 (IRAK4), MAP/microtubule affinity-regulating kinase 3 (MARK3), and TANK-binding kinase 1 (TBK1) were affected within 15 min of infection. Application of the corresponding kinase inhibitors of PKC, IRAK4, and p38 showed significant inhibition of SARS-CoV-2 replication. Additionally, proinflammatory cytokine production was reduced by applying PKC and p38 inhibitors. By an acquisition of a combined image data using positive- and negative-sense RNA probes, as well as pseudovirus entry assay, we demonstrated that PKC contributed to viral entry into the host cell, and therefore, could be a potential COVID-19 therapeutic target. Download figure Download PowerPoint Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has been identified to be the cause of coronavirus disease 2019 (COVID-19) since December 2019,1–3 posing huge challenges on health, social, and economic systems globally. SARS-CoV-2 is a zoonotic betacoronavirus; its exact origin or reservoir has not been defined.4–6 Besides, several betacoronaviruses have infected humans, causing respiratory diseases.7 SARS-CoV-2 infection could induce asymptomatic, mild to severe disease, characterized by a range of symptoms, including fever, dry cough, extreme tiredness associated with acute respiratory distress syndrome (ARDS), and lung injury.3,8 Presently, there are no clinically approved antiviral drugs that could effectively inhibit the replication of the SARS-CoV-2. It has been shown that SARS-CoV-2 has a structure similar to receptor-binding domain (RBD) like SARS-CoV or cellular receptor angiotensin-converting enzyme 2 (ACE2), critical for its entry into the host cell.9 Therefore, targeting the viral entry process could be a useful approach to antiviral strategy for COVID-19.10 Enfuvirtide was the first approved viral entry inhibitor that obstructed HIV fusion to host cells.11 Fusion inhibitors, blocking the fusion process of multiple viruses have been developed and shown antiviral activity in vivo.12,13 Consequently, gaining knowledge on SARS-CoV-2 viral entry stage is an urgent requirement for valuable drug development. It is widely reported that coordinated kinase activities are crucial during viral entry.14,15 For example, an activation of protein kinase C (PKC) contributed to influenza viral entry through late endosomes.14,15 Profiling of kinase activity post influenza virus infection showed G protein-coupled receptor kinase 2 was activated within 5 min of influenza infection.16 A comprehensive analysis on SARS-CoV-2 phosphorylation networks during viral uncoating to replication phase (2–24 h postinfection [PI]) was performed by Bouhaddou et al.,17 which demonstrated that SARS-CoV-2 infection promoted multiple kinases' activation, including casein kinase II and p38. However, information on how phosphorylation dynamics changes during the SARS-CoV-2 entry process is still limited. Results and Discussion To determine the appropriate time points to examine changes in the host phosphorylation network, transmission electron microscopy (TEM) was employed to monitor the cell entry process of SARS-CoV-2 within the first 30 min of host infection (Figure 1a). A fetal rhesus monkey kidney Vero E6 cell line was used as a model of infection, as it is highly susceptible to SARS-CoV-2. A high multiplicity of infection (MOI) of 25 was used to ensure universal infection of the Vero E6 cells. Viral particles were found to attach onto the cell surface at 5 min post viral infection, while a thickened membrane was observed at 15 min PI (Figure 1a), denoting the areas where the viral envelope was fusing with the plasma membrane. Within the first 30 min, whole virion was no longer observed. Instead, viral cores of consistent size (40–50 nm in diameter) without envelope were observed in large vacuoles (Figure 1a, 30 min), indicative of the endosome, as coronavirus entered the cells by endocytosis.18 Hence, we selected 5 and 15 min PI as time points to examine the phosphorylation dynamics during viral infection. Figure 1 | Phosphorylation network response during the early phase of SARS-CoV-2 infection. (a) TEM analysis of Vero E6 infected with SARS-CoV-2 for 5, 15, 30 min. Viral particles attaching and fusing with cell membrane (5 and 15 min, black arrow). Some viral particles were observed in the large vacuoles (30 min, white arrowhead). (b) Workflow of phosphopeptides' enrichment. (c and d) Regulation of phosphopeptides identified in 5 (c) and 15 min (d) PI with SARS-CoV-2. Download figure Download PowerPoint Vero E6 cells were harvested at 5 or 15 min PI after SARS-CoV-2 or mock infection (Figure 1b) in four independent biological experiments. At 5 min PI, 115 phosphorylation sites were upregulated and 106 were downregulated, while 37 were upregulated and 65 were downregulated at 15 min PI (Figures 1c and 1d). The altered phosphopeptides were then used for further functional analysis and kinase prediction. At both study time-points, PKC activity was the highest enriched molecular function by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis ( Supporting Information Figure S1a). Enrichment map analysis also showed significant overlaps in PKC related pathways ( Supporting Information Figure S1c), suggesting that kinase activities were perturbed during the viral entry process. We then established a kinase prediction pipeline using altered phosphosites (both up- and down-altered) identified. Kinase prediction was obtained initially by the Group-based Prediction System 5.0,19,20 followed by highly stringent cutoff adopted from previous publication.16 Four kinases were consistently predicted at both 5 and 15 min PI (Figure 2a), and consequently selected for further validation, as follows: PKC-gamma (PKCγ; p = 0.0109, which was also identified directly in phosphoproteomics analysis), interleukin-1 receptor-associated kinase 4 (IRAK4; p = 0.0203), MAP/microtubule affinity-regulating kinase 3 (MARK3; p < 0.0001), and TANK-binding kinase 1 (TBK1; p = 0.0003). Subsequently, amino acid sequences flanking hyperphosphorylation sites were retrieved to reveal the phosphorylation motifs (Figure 2b). Basophilic motif of arginine (R) at position-3 was significantly enriched, compared with background, a specific feature of conventional PKCs, including PKCγ,21 which was in agreement with our prediction. Intriguingly, recent studies showed that MARK3 and TBK1 could directly interact with SARS-CoV-2 viral proteins.22 Furthermore, TBK1 was targeted by SARS-CoV-2 proteins to antagonize type I interferon (IFN-I) response.23 Collectively, these lines of evidence confirmed our kinase prediction pipeline was able to identify crucial kinase for SARS-CoV-2 replication (Figure 2c). Figure 2 | Predicted early responsive kinase activity of SARS-CoV-2 infection. (a) Top kinases predicted to regulate differential phosphorylation at 5- and 15-min PI are marked in red. All enriched kinases passed the stringent score filter. (b) Enriched phosphorylation motifs from hyperphosphorylated peptides in both 5 and 15 min PI, phosphorylated sites (S/T) set as position 0. Size of the letter represents the enrichment degree. (c) Protein–protein interactions (PPI) map of SARS-CoV-2 viral protein with predicted host kinase-substrates network. Download figure Download PowerPoint Further, we used kinase inhibitors targeting the predicted kinases to evaluate their effects on viral replication. Inhibitors of PKC (Bisindolylmaleimide IX), TBK1 (Amlexanox & MRT67307 HCL), and IRAK (IRAK-1-4 Inhibitor I) were used. p38 kinase MAPK12 and cyclin-dependent kinase 6 (CDK6) were top-predicted kinases before a stringent filter was applied, and the hyperphosphorylated motifs suggested the involvement of CDK and MAPK kinases (Figure 2b). In addition, p38 was reported to affect SARS-CoV replication.24 Therefore, we included CDK inhibitor (Palbociclib HCI) and p38 inhibitor (SB203580) as well. Cytotoxicity of these inhibitors were determined ( Supporting Information Figure S2). Then inhibitors were used at concentrations without major cytotoxicity. Bafilomycin A1 (BafA1), reported to block SARS-CoV-2 viral entry,25,26 was used as positive control. As shown in Figure 3a, p38 inhibitor efficiently blocked virus replication in all three cell lines, as expected. Inhibition of either IRAK or PKC leads to suppression of viral replication and viral mRNA synthesis in a dose-dependent manner in both Calu3 (non-small-cell lung cancer) and Caco2 (human colorectal adenocarcinoma) cell lines (Figure 3b), confirming our kinase prediction. Importantly, PKC the inhibitor showed the most pronounced inhibition of viral replication and mRNA synthesis, consistent with both KEGG (Kyoto Encyclopedia of Genes and Genomes, a database resource for functional studies) analysis and kinase prediction. As kinase inhibitors might have off-target effect, two additional PKC inhibitors (Sotrastaurin and Enzastaumn) were used to evaluate their effects on viral replication ( Supporting Information Figure S5). All three PKC inhibitors demonstrated inhibitory effects on SARS-CoV-2 replication in a dose-dependent manner, confirming the critical role of PKC activity in viral replication. The discrepancy of inhibitory effects for kinase inhibitors were observed between Vero E6 and two human cell lines, which should be caused by the absence of IFN-I in cells ( Supporting Information Figure S3). The lack of IFN-I would only affect the overall viral replication but not the phosphodynamics we observed, as we focused on the viral entry process, which was before the participation of IFN-I. This observation also confirmed the role of interferon, as reported previously.27 Inhibition of CDK led to a slight increase in terms of viral replication, suggesting that alteration of CDK kinase activity might be an adversary for SARS-CoV-2 replication. Figure 3 | Effect of different inhibitor treatment on viral mRNA level and viral titer. Cells were pretreated with different inhibitors at the indicated dose, followed by SARS-CoV-2 infection. Indicated three different relative viral mRNA levels cells were measured by normalizing to control (a). Corresponding viral titers by plaque assay were shown in (b). For all panels, *p < 0.05, **p < 0.005, ***p < 0.0005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Furthermore, we used RNA fluorescence in situ hybridization (RNA-FISH) to visualize the viral replication process. SARS-CoV-2 generates negative-strand RNA template to synthesize new genomic RNAs; therefore, the distribution of negative-strand RNA refers to the location of replicative-intermediate in replication-transcription complex.28 An A549 cell line expressing human ACE2 was generated (Figure 4c) and pretreated with inhibitors before SARS-CoV-2 infection. In the control group, viral genomic RNA and mRNA were widely distributed and accumulated in the perinuclear area. Replicative-intermediate RNAs, indicative of ongoing viral replication, were also clearly detected (Figure 4). In contrast, p38 or PKC inhibitors significantly repressed viral infection rate. Particularly, effect of PKC inhibitor was more significant than BafA1-positive control (Figure 4a). We found that inhibitor of p38 kinase did not change signal intensity of negative-sense viral RNA, suggesting the inhibition probably occurred in the late stage of viral cycle. However, perinuclear dots of negative-sense viral RNA were significantly decreased after the treatment of PKC inhibitor (Figure 4b), which signified the lack of ongoing replication events. The observation indicated that PKC activity was crucial during viral entry as we predicted. We then validated the role of PKC during the early stage of viral replication by SARS-CoV-2 pseudovirus entry assay. A home-made SARS-CoV-2 pseudovirus was constructed and used to infect 293T-ACE2 cells pretreated with PKC inhibitors. As shown in Figure 4d, an inhibition of PKC activity diminished pseudovirus signals, confirming the inhibitory role of PKC inhibitor during the early stage of SARS-CoV-2 replication. PKC is known to regulate PKC-dependent endocytosis and involve in influenza viral entry by regulating late endosomes.14,15 Alternatively, coronaviruses, including Middle East respiratory syndrome (MERS)- and SARS-CoV, are known to rely on endocytic pathway for entry.18,29 Consequently, we proposed that PKC was required by SARS-CoV-2 as an early responsive kinase for viral entry via an endocytic pathway, making it a potential therapeutic target for COVID-19. Figure 4 | Confocal images suggested that PKC inhibitor block SARS-CoV-2 entry. A549-Ace2 cells were pretreated with the indicated inhibitor, followed by SARS-CoV-2 infection. (a) FISH and IFA imaging of infected cells using positive-sense RNA probe (purple) and antibody against viral N protein (green). (b) Fixed cells were processed for FISH assay using positive- (purple) and negative-sense RNA probe (green). Merge images also include 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (c) Whole cell lysates were analyzed for Ace2 and tubulin expression by Western blot using their respective antibodies. (d) Pseudovirus entry assay showed that the inhibition of PKC activity prevented viral entry signals. Download figure Download PowerPoint It has been reported that poor prognosis outcomes of patients with COVID-19 were associated with cytokine storm, generated by innate immune response, while several cytokines have been reported as potential biomarkers for disease progression.30–32 Additionally, emerging pieces of evidence have shown that SARS-CoV-2 infection induces low types I and III IFNs' levels and limited interferon-stimulated genes' (ISG) response, but high level of chemokine expression.8,33 We showed that the inhibition of cytokine mRNA levels caused by kinase inhibitors correlated with that of viral titer. A significant and dose-dependent reduction of cytokine levels were observed when treated with PKC and p38 inhibitors across all three cell lines ( Supporting Information Figure S4). Also, these cytokines' expression were inhibited by IRAK inhibitor, but at a relatively moderate level. Finally, to validate the essential role of PKC activities in viral replication, three commercially available small interfering RNAs (siRNAs) targeting PKC-alpha (PKCα), PKC-beta (PKCβ), and PKC-epsilon (PKCɛ) were ordered to knock down the corresponding PKC isoforms. PKCα siRNA failed to achieve effective knock down, so only PKCβ and PKCɛ siRNAs were used in viral inhibition assay (Figure 5a). As shown in Figure 5b, only PCKβ knock down showed significant inhibitory effect on viral replication. A siRNA knock down of PKCɛ led to an increase in PKCβ activity, suggesting a potential compensation effect, which might explain the inefficiency of viral inhibition of PKCɛ siRNA. These results further confirmed with our earlier PKC inhibitors' data, as all three PKC inhibitors we tested in the study are efficient PKCβ inhibitors. Consequently, PKC activity, particularly PKCβ, might play a vital role in optimum replication of SARS-CoV-2. Figure 5 | siRNA knock down of PKCβ inhibits SARS-CoV-2 replication. (a) Transcriptional levels of PKC isoforms in siRNA knock down Calu-3 cells were examined using q-PCR. (b) Viral mRNA levels in siRNA knock down Calu-3 cells infected with SARS-CoV-2 were measured by normalizing to control. **p < 0.005, ***p < 0.0005, ****p < 0.00005, nonsignificant (ns) for two-tail Student's t-test. Error bars indicate SD (n = 3). Download figure Download PowerPoint Conclusion Using a time-course TEM imaging, we identified key time points of viral attachment and fusion of SARS-CoV-2 infection. By combining phosphoproteomics and kinase prediction pipeline, we found that PKC and IRAK4 activities were activated at the first 5–15 min of viral entry. We showed that the inhibition of PKC, IRAK4, and p38 could suppress optimal replication of the SARS-CoV-2 virus, among which IRAK4 activity initially associated with SARS-CoV-2 replication. We further demonstrated that inhibition of PKC activity, particularly PKCβ, would inhibit viral replication at early stage, probably via blockage of specific endocytosis phosphorylation events required for viral entry. Therefore, PKC might be required for SARS-CoV-2 entry, and thus, could serve as a potential therapeutic target for COVID-19. Data Availability The raw MS data from this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with accession number PXD021610. Supporting Information Supporting Information is available and includes detailed material and methods, as well as Figures S1–S5. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible because of a generous grant from the National Key R&D Program, Ministry of Science and Technology, China (no. 2017YFC1600500), the National Natural Science Foundation of China (no. 21705137), the Theme-Based Research Scheme (no. T11/707/15) and General Research Fund (no. 17107019) of the Research Grants Council, Hong Kong Special Administrative Region, and the Sanming-Project of Medicine in Shenzhen, China (nos. SZSM201911014 and SZSM201811070).

Headful DNA packaging: Bacteriophage SPP1 as a model system

Accurate classification of bacteriophage proteins (also known as phage proteins) is crucial for understanding their functions, particularly for key proteins of Caudovirales phages such as the portal protein, the large terminase subunit (TerL), and the small terminase subunit (TerS). To address the challenges in identifying these proteins, we propose a novel method named CNN-SANs, which integrates Convolutional Neural Networks (CNN) with Self-Attention Mechanisms (SANs). Our approach uses one-hot encoded protein sequence data as input, where CNNs extract local features through convolutional and pooling layers to identify key patterns. Subsequently, self-attention mechanisms dynamically adjust the focus on different positions within the sequences to capture long-range dependencies and global context. Experimental results demonstrate that applying CNN-SANs to metagenomic data significantly enhances Precision and F1 Score in protein identification. Compared to traditional methods, CNN-SANs exhibits superior performance in accurately classifying Caudovirales phage proteins, proving its effectiveness as a powerful tool for bacteriophage identification.

ROLE OF THE SMALL TERMINASE SUBUNIT ENCODED BY STAPHYLOCOCCUS AUREUS PATHOGENICITY ISLAND SAPI1 IN FORMATION OF SAPI1 TRANSDUCING PARTICLES By Nicholas Paul Olivarez, M.S. A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2008 Major Director: Dr. Gail E Christie Professor, Microbiology & Immunology Staphylococcus aureus pathogenicity island SaPI1 is a genomic element that is mobilized and transduced at high frequency by helper phage 80α. SaPI1 encodes a small terminase protein that belongs to the phage small terminase subunit family. The presence of SaPI1encoded small terminase suggests that it plays a role in SaPI1-specific packaging into transducing particles by complexing with the 80α large terminase subunit and redirecting recognition to a pac site on SaPI1 DNA from 80α DNA. The effects of deleting the small xiii terminase genes in SaPI1 and in a prophage copy of 80α are consistent with this hypothesis. Induction of the 80α small terminase deletion mutant produces wild type levels of SaPI1 transducing particles, demonstrating that SaPI1 small terminase can replace that of 80α in SaPI1 packaging. Southern blot analysis of virion DNAs isolated from the deletion mutants confirms that SaPI1 redirects packaging of its DNA into SaPI1-sized capsids. CHAPTER

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

BackgroundBurkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China.MethodsBurkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test.ResultsA phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3–12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage.ConclusionsAs the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.Graphical