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Journal of Virology Apr 2016Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human...
UNLABELLED
Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen herpes simplex virus 1 (HSV-1) and the animal herpesvirus pseudorabies virus (PRV), encode a membrane-bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed the visualization of viral infections by microscopy and flow cytometry as well as the differentiation of viral progeny. Our results demonstrated the majority of HSV-1- and PRV-infected cells establish superinfection exclusion by 2 h postinfection. The modification of viral infections by virion inactivation and phosphonoacetic acid, cycloheximide, and actinomycin D treatments indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of a secondary viral inoculum. We also identified the timing of coinfection events during axon-to-cell spread, with most occurring within a 2-h window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during the pathogenic spread of infection.
IMPORTANCE
Superinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating the development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to coinfect the same cell and generate viral recombinants will enhance the development of effective antiviral therapies and interventions.
Topics: Animals; Cell Line; Cells, Cultured; Chlorocebus aethiops; Herpesvirus 1, Human; Herpesvirus 1, Suid; Rats; Reassortant Viruses; Superinfection; Vero Cells; Viral Envelope Proteins
PubMed: 26842480
DOI: 10.1128/JVI.00089-16 -
Journal of Clinical Virology : the... Oct 2023Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have...
BACKGROUND
Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric.
METHODS
We used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing.
RESULTS
Among control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA.
CONCLUSIONS
These data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.
Topics: Humans; Antiviral Agents; Herpesvirus 2, Human; Acyclovir; Foscarnet; Herpesvirus 1, Human; Genotype; Drug Resistance, Viral; Herpes Simplex
PubMed: 37586184
DOI: 10.1016/j.jcv.2023.105554 -
Frontiers in Cellular and Infection... 2017Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of...
Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of phosphonates in the marine biosphere is well characterized but the role of these molecules in the intestine is poorly understood. uses its virulence factors to influence the host immune response to compete with the host and normal microflora for nutrients. cannot produce phosphonates but encodes the enzymes to use them suggesting that it is exposed to phosphonates during its life cycle. The role of phosphonates during enteric salmonellosis is unexplored. We have previously shown that , encoding a putative regulator of phosphonate metabolism, is needed for colonization in calves. Here, we report that the necessity of in colonization of the murine intestine results from multiple factors. is needed for full activation of the type-3 secretion system-1 and for optimal invasion of epithelial cells. The Δ mutant grows poorly in phosphonoacetic acid (PA) as the sole phosphorus source, but can use 2-aminoethylphosphonate. PhnA, an enzyme required for PA breakdown, is not controlled by STM3602 suggesting an additional mechanism for utilization of PA in . Typhimurium. Finally, the requirement of for intestinal colonization differs depending on the composition of the microflora. Our data suggest that has multiple regulatory targets that are necessary for survival within the microbial community in the intestine. Determination of the members of the regulon may illuminate new pathways needed for colonization of the host.
Topics: Animals; Gene Expression Regulation, Bacterial; Intestines; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Phosphonoacetic Acid; Salmonella Infections, Animal; Salmonella enterica
PubMed: 28361036
DOI: 10.3389/fcimb.2017.00069 -
ACS Chemical Biology Oct 2017Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated...
Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a D*A transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.
Topics: ATP Phosphoribosyltransferase; Bacteria; Bacterial Proteins; Binding Sites; Catalytic Domain; Kinetics; Models, Molecular; Protein Conformation; Protein Isoforms; Signal Transduction
PubMed: 28872824
DOI: 10.1021/acschembio.7b00484 -
ACS Synthetic Biology Feb 2017The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the...
The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.
Topics: Biological Products; Hydrolases; Multigene Family; Phosphonoacetic Acid; Phosphorous Acids; Streptomyces; Synthetic Biology
PubMed: 28103011
DOI: 10.1021/acssynbio.6b00299 -
Antimicrobial Agents and Chemotherapy Feb 2021The objectives of this study were to characterize the role of the , , and genes in fosfomycin resistance in and evaluate the use of sodium phosphonoformate (PPF) in...
The objectives of this study were to characterize the role of the , , and genes in fosfomycin resistance in and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of and the reference strain (ATCC 700721) were used, and their genomes were sequenced. Δ, Δ, and Δ mutants were constructed from two isolates and ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512 mg/liter, activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307 mg/liter were evaluated with and without PPF (0.623 mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024 mg/liter. The addition of 0.623 mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623 mM). The lowest fosfomycin-resistant mutant frequencies were found in Δ mutants, with decreases in frequency from 1.69 × 10 to 1.60 × 10 for 64 mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of and not with the addition of PPF. We conclude that gene inactivation leads to a decrease in fosfomycin resistance in The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations.
Topics: Anti-Bacterial Agents; Foscarnet; Fosfomycin; Klebsiella pneumoniae; Microbial Sensitivity Tests; beta-Lactamases
PubMed: 33361305
DOI: 10.1128/AAC.01911-20 -
Journal of Enzyme Inhibition and... Dec 2018Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity...
Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.
Topics: DNA-Directed DNA Polymerase; Enzyme Inhibitors; HIV-1; Molecular Structure; Moloney murine leukemia virus; Nucleotides; Phosphonoacetic Acid
PubMed: 29372656
DOI: 10.1080/14756366.2017.1417275 -
Journal of Virology Jul 2017Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B...
Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including , , and , without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state. The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.
Topics: Cell Culture Techniques; Cell Line; Herpesvirus 6, Human; Humans; In Situ Hybridization, Fluorescence; Real-Time Polymerase Chain Reaction; Virus Cultivation; Virus Integration
PubMed: 28468878
DOI: 10.1128/JVI.00437-17 -
ACS Biomaterials Science & Engineering Apr 2022[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a...
[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO][(FCN)(OH)] and Gd[FCN] IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.
Topics: Antiviral Agents; Cytomegalovirus; Foscarnet; Herpesvirus 1, Human; Humans; Nanoparticles
PubMed: 35344659
DOI: 10.1021/acsbiomaterials.2c00074 -
Journal of Nanobiotechnology Jul 2022Cytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune...
BACKGROUND
Cytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune Deficiency Syndrome patients. Antiviral drugs ganciclovir (GCV) and phosphonoformate (PFA) are first-line agents for pneumonia caused by herpesvirus infection. However, the therapy suffers from various limitations such as low efficiency, drug resistance, toxicity, and lack of specificity.
METHODS
The antiviral drugs GCV and PFA were loaded into the pH-responsive nanoparticles fabricated by poly(lactic-co-glycolic acid) (PLGA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and further coated with cell membranes derived from bone marrow mesenchymal stem cells to form artificial stem cells, namely MPDGP. We evaluated the viral suppression effects of MPDGP in vitro and in vivo.
RESULTS
MPDGP showed significant inflammation tropism and efficient suppression of viral replication and virus infection-associated inflammation in the CMV-induced pneumonia model. The synergistic effects of the combination of viral DNA elongation inhibitor GCV and viral DNA polymerase inhibitor PFA on suppressing the inflammation efficiently.
CONCLUSION
The present study develops a novel therapeutic intervention using artificial stem cells to deliver antiviral drugs at inflammatory sites, which shows great potential for the targeted treatment of pneumonia. To our best knowledge, we are the first to fabricate this kind of artificial stem cell to deliver antiviral drugs for pneumonia treatment.
Topics: Antiviral Agents; Cytomegalovirus; Cytomegalovirus Infections; Fatty Acids, Monounsaturated; Foscarnet; Ganciclovir; Humans; Inflammation; Nanoparticle Drug Delivery System; Pneumonia; Polylactic Acid-Polyglycolic Acid Copolymer; Quaternary Ammonium Compounds; Stem Cells
PubMed: 35842662
DOI: 10.1186/s12951-022-01547-x