RESUMEN
Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.
Asunto(s)
Alphaherpesvirinae , Herpesviridae , Enfermedad de Marek , Animales , Proteínas Quinasas/metabolismo , Cromatografía Liquida , Pollos , Espectrometría de Masas en Tándem , Herpesviridae/metabolismo , Alphaherpesvirinae/metabolismo , Proteínas Virales/metabolismo , Virus OncogénicosRESUMEN
Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.
Asunto(s)
Herpesviridae , Herpesvirus Gallináceo 2 , Enfermedad de Marek , Proteogenómica , Humanos , Animales , Pollos , Herpesviridae/metabolismo , Herpesvirus Gallináceo 2/genéticaRESUMEN
Natural pigments are becoming increasingly popular owing of their reliability. Microbial pigments provide an alternative to natural colours. A total of 24 fungal cultures were collected from leaf bits of Senna auriculata, with one strain (FNG1) producing an extracellular red orange pigment. Nigrospora oryzae was confirmed by using physical criteria and molecular phylogenetic study by using ITS and ß- tubulin analysis. In EtOAc, the crude red pigment was the most soluble. The TLC analysis was used to partly purify the natural pigment. The partially purified fungal pigment was used in successive bioprospecting studies. The antimicrobial activity of the partially purified sample was assessed against eight human pathogens, with Leucobacter AA7 showing the largest zone of inhibition (200-500 µg/mL). The compound's DPPH scavenging activity enhanced from 38.2 to 67.9%, with an IC50 value of 34.195 ± 2.33 µg/mL. Cancer cells were suppressed by partly pure fungal pigment, but non-cancerous HEK 293 cells were unaffected. The GC-MS analysis was used to characterize the molecule present in the partly purified pigment. In addition, the cotton textiles have the greatest staining capability for crude mycobial pigment, which dyes quickly and has a negative cytotoxicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01211-y.
RESUMEN
The Herpesviridae encode many conserved genes, including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture to various degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek's disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken to chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and Western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread and autophosphorylation of CHPK and that mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens, and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread.IMPORTANCE Marek's disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek's disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation but do not block interindividual spread from chicken to chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek's disease while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined that MDV CHPK is not required for cell-to-cell spread, for disease induction, and for oncogenicity. However, it is required for interindividual spread, and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.
Asunto(s)
Alphaherpesvirinae/metabolismo , Pollos/virología , Herpesviridae/metabolismo , Enfermedad de Marek/virología , Proteínas Quinasas/metabolismo , Proteínas Virales/metabolismo , Animales , Epítopos , Herpesvirus Gallináceo 2 , Enfermedad de Marek/transmisión , Modelos Moleculares , Enfermedades de las Aves de Corral/virología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Piel/patología , Piel/virología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.
Asunto(s)
Herpesviridae/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Alphaherpesvirinae/genética , Animales , Pollos/virología , Genes Virales , Herpesviridae/genética , Herpesviridae/patogenicidad , Infecciones por Herpesviridae/metabolismo , Proteínas Inmediatas-Precoces/fisiología , Enfermedad de Marek/genética , Enfermedad de Marek/virología , Aves de Corral/virología , Proteínas ViralesRESUMEN
Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (EGSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (ß-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and ß-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1-independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of ß-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while ß-lap elicits off-target responses at its effective doses.
Asunto(s)
Antineoplásicos/farmacología , Glutatión/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Estrés Oxidativo/efectos de los fármacos , Quinonas/farmacología , Técnicas Biosensibles , Línea Celular Tumoral , Citosol/efectos de los fármacos , Citosol/metabolismo , Dicumarol/farmacología , Colorantes Fluorescentes/análisis , Glutarredoxinas/análisis , Glutarredoxinas/genética , Glutatión/análisis , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Indolquinonas/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Imagen Molecular , Sondas Moleculares/genética , Terapia Molecular Dirigida , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , Naftoquinonas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Especificidad por SustratoRESUMEN
Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.
Asunto(s)
Antioxidantes/farmacología , Complejo III de Transporte de Electrones/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , Animales , Técnicas Biosensibles , Células CHO , Cricetulus , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Células HCT116 , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Porcinos , Factores de Tiempo , TransfecciónRESUMEN
Oncogenic viruses play a pivotal role in oncology due to their unique role in unraveling the complexities of cancer development. Understanding the role viruses play in specific cancers is important to provide basic insights into the transformation process, which will help identify potential cellular targets for treatment. This review discusses the diverse role of animal herpesviruses in initiating and promoting various forms of cancer. We will summarize the mechanisms that underlie the development of animal herpesvirus-induced cancer that may provide a basis for developing potential therapeutic interventions or preventative strategies in the future.
Asunto(s)
Infecciones por Herpesviridae , Herpesviridae , Neoplasias , Virus Oncogénicos , Animales , Herpesviridae/fisiología , Herpesviridae/patogenicidad , Herpesviridae/genética , Humanos , Neoplasias/virología , Infecciones por Herpesviridae/virología , Virus Oncogénicos/fisiología , CarcinogénesisRESUMEN
Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens causing pulmonary infection to fatal disseminated disease. NTM infections are steadily increasing in children and adults, and immune-compromised individuals are at a greater risk of fatal infections. The NTM disease's adverse pathology and resistance to antibiotics have further worsened the therapeutic measures. Innate immune regulators are potential targets for therapeutics to NTM, especially in a T cell-suppressed population, and many ubiquitin ligases modulate pathogenesis and innate immunity during infections, including mycobacterial infections. Here, we investigated the role of an E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (CBLB), in immunocompromised mouse models of NTM infection. We found that CBLB is essential to prevent bacterial growth and dissemination. Cblb deficiency debilitated natural killer cells, inflammatory monocytes, and macrophages in vivo. However, Cblb deficiency in macrophages did not wane its ability to inhibit bacterial growth or production of reactive oxygen species or interferon γ production by natural killer cells in vitro. CBLB restricted NTM growth and dissemination by promoting early granuloma formation in vivo. Our study shows that CBLB bolsters innate immune responses and helps prevent the dissemination of NTM during compromised T cell immunity.
Asunto(s)
Inmunidad Innata , Infecciones por Mycobacterium no Tuberculosas , Proteínas Proto-Oncogénicas c-cbl , Animales , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Ratones , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Células Asesinas Naturales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Micobacterias no Tuberculosas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Granuloma/inmunología , Granuloma/microbiología , Granuloma/patologíaRESUMEN
Purinergic receptors (PRs) have been reported as potential therapeutic targets for many viral infections including herpesviruses, which urges the investigation into their role in Marek's disease (MD), a herpesvirus induced cancer in chickens that is an important pathogen for the poultry industry. MD is caused by MD virus (MDV) that has a similar viral life cycle as human varicella zoster virus in that it is shed from infected epithelial skin cells and enters the host through the respiratory route. In this report, PR responses during natural MDV infection and disease progression was examined in MD-resistant white Leghorns (WL) and MD-susceptible Pure Columbian (PC) chickens during natural infection. Whole lung lavage cells (WLLC) and liver tissue samples were collected from chickens infected but showing no clinical signs of MD (Infected) or presenting with clinical disease (Diseased). RNA was extracted followed by RT-qPCR analysis with gene specific primers against members of the P1, P2X, and P2Y PR families. Differential expression (p < 0.05) was observed in breed and disease conditions. Some PRs showed tissue specific expression (P1A1, P2X1, and P2X6 in WLLC) whereas others responded to MDV infection only in MD-susceptible (PC) chickens (P1A2A, P2X1, P2X5, P2X7). P2Y PRs had differential expression in both chicken lines in response to MDV infection and MD progression. This study is the first to our knowledge to examine PR responses during MDV infection and disease progression. These results suggest PR signaling may an important area of research for MDV replication and MD.
Asunto(s)
Herpesviridae , Herpesvirus Gallináceo 2 , Enfermedad de Marek , Animales , Humanos , Pollos/genética , Herpesviridae/metabolismo , Herpesvirus Gallináceo 2/genética , Susceptibilidad a Enfermedades , Progresión de la EnfermedadRESUMEN
One of the greatest achievements of the last century is the development of vaccines against viral diseases. Vaccines are essential for battling infectious diseases and many different formulations are available, including live attenuated vaccines. However, the use of live attenuated vaccines has the potential for adverse effects, including reversion of pathogenicity, recombination, and functional complementation in the host. Marek's disease is a serious disease in poultry controlled by live attenuated vaccines that has resulted in increased virulence over the decades. Recombination between circulating field viruses or vaccines is a proposed mechanism for the increase in virulence, however, complementation between vaccines and field strains has not been demonstrated in chickens. Here, we describe functional complementation of vaccines with virulent virus to functionally complement transmission and spread in the host. Using the natural virus-host model of Marek's disease in chickens, our results show dual infection of target cells in chickens with vaccine and virulent virus providing the opportunity for recombination or complementation to transpire. Interestingly, our controlled results showed no evidence of recombination between vaccine and virulent virus, but functional complementation occurred in two independent experiments providing proof for complementation during natural infection in vaccinated individuals. These results suggest complementation as a potential mechanism for vaccine-mediated viral evolution and the potential for complementation should be taken into consideration when developing novel vaccines.
Asunto(s)
Coinfección , Enfermedad de Marek , Enfermedades de las Aves de Corral , Vacunas Virales , Virus , Animales , Pollos , Enfermedad de Marek/prevención & control , Vacunas Atenuadas/genética , Vacunas Virales/genéticaRESUMEN
Marek's disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus (Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus (Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera (Mardivirus vs. Iltovirus) and not the host (Gallid vs. Meleagrid).
Asunto(s)
Antígenos Virales/metabolismo , Pollos/virología , Herpesvirus Gallináceo 2/fisiología , Enfermedad de Marek/transmisión , Enfermedad de Marek/virología , Proteínas del Envoltorio Viral/metabolismo , Animales , Antígenos Virales/genética , Células Cultivadas , Herpesvirus Gallináceo 1/clasificación , Herpesvirus Gallináceo 1/genética , Herpesvirus Meleágrido 1/clasificación , Herpesvirus Meleágrido 1/genética , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Proteínas Recombinantes/metabolismo , Pavos/virología , Proteínas del Envoltorio Viral/genética , Replicación ViralRESUMEN
We have formerly shown that glycoprotein C (gC) of Gallid alphaherpesvirus 2, better known as Marek's disease (MD) alphaherpesvirus (MDV), is required for interindividual spread in chickens. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized gC was important for interindividual spread of other alphaherpesviruses. To test this hypothesis, we first generated a fluorescent protein tagged clone of Gallid alphaherpesvirus 3 MD vaccine strain 301B/1 to track virus replication in cell culture and chickens using fluorescent microscopy. Following validation of this system, we removed the open reading frame of 301B/1 gC from the genome and determined whether it was required for interindividual spread using experimental and natural infection studies. Interindividual spread of MD vaccine 301B/1 was abrogated by removal of 301B/1 gC. Rescuent virus in which 301B/1 gC was inserted back into the genome efficiently spread among chickens. To further study the conserved function of gC, we replaced 301B/1 gC with MDV gC and this virus also efficiently spread in chickens. These data suggest the essential function of alphaherpesvirus gC proteins is conserved and can be exploited during the generation of future vaccines against MD that affects the poultry industry worldwide.
Asunto(s)
Pollos/virología , Herpesvirus Gallináceo 2/patogenicidad , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Animales , Herpesvirus Gallináceo 2/metabolismo , Herpesvirus Gallináceo 2/fisiología , Enfermedad de Marek/transmisión , Enfermedad de Marek/virología , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/química , Replicación ViralRESUMEN
Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that causes Marek's disease (MD) in chickens. RLORF4 is a MDV-specific gene that is systematically deleted during attenuation of MDV in vitro. Concomitantly, the expression of glycoprotein C (gC) is diminished during attenuation, suggesting these two changes may be linked. Original studies in which RLORF4 was deleted utilized an infectious clone that lacked gC expression due to a frame-shift mutation within the gC open reading frame (UL44). Here, we utilized an infectious clone in which gC expression was restored to test our hypothesis that RLORF4 is important for expression of MDV gC, and subsequently, interindividual spread. Contrary to our hypothesis, gC expression was unaltered during both in vitro and in vivo replication of RLORF4-null MDV and was able to efficiently transmit from chicken to chicken, conclusively showing that RLORF4 does not regulate gC expression and is not required for horizontal transmission.