RESUMO
With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , RNA/genética , Urina/microbiologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Células Cultivadas , Eritrócitos/parasitologia , Fungos/classificação , Fungos/efeitos dos fármacos , Fungos/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/genética , Humanos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.
Assuntos
Cátions Bivalentes/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Sequência Conservada , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Alinhamento de Sequência , Células Vero , Proteínas Virais/genéticaRESUMO
Many viruses regulate gene expression, both globally and specifically, to achieve maximal rates of replication. During herpes simplex virus 1 infection, translation of the DNA polymerase (Pol) catalytic subunit is inefficient relative to other proteins of the same temporal class (D. R. Yager, A. I. Marcy, and D. M. Coen., J. Virol. 64:2217-2225, 1990). To investigate the mechanisms involved in the inefficient translation of Pol and to determine whether this inefficient translation could affect viral replication, we performed a mutagenic analysis of the 5' end of the pol transcript. We found that a short sequence ( approximately 55 bases) in the 5' leader of the transcript is both necessary and sufficient to inhibit translation in rabbit reticulocyte lysates and sufficient to inhibit reporter gene translation in transfected cells. RNase structure mapping experiments indicated that the inhibitory element adopts a structure that contains regions of a double-stranded nature, which may interfere with ribosomal loading and/or scanning. Pol accumulated to approximately 2- to 3-fold-higher levels per mRNA in cells infected with a mutant virus containing a deletion of the approximately 55-base inhibitory element than in cells infected with a control virus containing this element. Additionally, the mutant virus replicated less efficiently than the control virus. These results suggest that the inhibitory element regulates Pol translation during infection and that its inhibition of Pol translation is beneficial for viral replication.
Assuntos
Regiões 5' não Traduzidas/fisiologia , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Herpesvirus Humano 1/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Virais/genética , Regiões 5' não Traduzidas/genética , Animais , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Genes Reporter , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Mutagênese , Conformação de Ácido Nucleico , RNA de Cadeia Dupla , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Deleção de Sequência , Células Vero , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.
Assuntos
Herpesvirus Humano 1 , Proteínas de Ligação a RNA/metabolismo , RNA/química , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Ensaio de Desvio de Mobilidade Eletroforética , Radical Hidroxila/química , Dados de Sequência Molecular , Oligonucleotídeos/química , Estrutura Terciária de Proteína , RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Ribonucleases/metabolismo , Proteínas Virais/químicaRESUMO
UNLABELLED: Herpes simplex virus (HSV) utilizes and subverts host chromatin mechanisms to express its lytic gene products in mammalian cells. The host cell attempts to silence the incoming viral genome by epigenetic mechanisms, but the viral VP16 and ICP0 proteins promote active chromatin on the viral genome by recruiting other host epigenetic factors. However, the dependence on VP16 and ICP0 differs in different cell lines, implying cell type-dependent functional contributions of epigenetic factors for HSV gene expression. In this study, we performed a targeted RNA interference (RNAi) screen for cellular chromatin factors that are involved in regulation of herpes simplex virus (HSV) gene expression in U2OS osteosarcoma cells, a cell line that complements ICP0 mutant and VP16 mutant virus replication. In this screen, we found the same general classes of chromatin factors that regulate HSV gene expression in U2OS cells as in other cell types, including histone demethylases (HDMs), histone deacetylases (HDACs), histone acetyltransferases (HATs), and chromatin-remodeling factors, but the specific factors within these classes are different from those identified previously for other cell types. For example, KDM3A and KDM1A (LSD1) both demethylate mono- and dimethylated H3K9, but KDM3A emerged in our screen of U2OS cells. Further, small interfering RNA (siRNA) and inhibitor studies support the idea that KDM1A is more critical in HeLa cells, as observed previously, while KDM3A is more critical in U2OS cells. These results argue that different cellular chromatin factors are critical in different cell lines to carry out the positive and negative epigenetic effects exerted on the HSV genome. IMPORTANCE: Upon entry into the host cell nucleus, the herpes simplex virus genome is subjected to host epigenetic silencing mechanisms. Viral proteins recruit cellular epigenetic activator proteins to reverse and counter the cellular silencing mechanisms. Some of the host silencing and activator functions involved in HSV gene expression have been identified, but there have been indications that the host cell factors may vary in different cell types. In this study, we performed a screen of chromatin factors involved in HSV gene regulation in osteosarcoma cells, and we found that the chromatin factors that are critical for HSV gene expression in these cells are different from those for previously studied cell types. These results argue that the specific chromatin factors operative in different cell lines and cell types may differ. This has implications for epigenetic drugs that are under development.
Assuntos
Epigênese Genética , Regulação Viral da Expressão Gênica , Testes Genéticos/métodos , Interações Hospedeiro-Patógeno , Simplexvirus/genética , Linhagem Celular Tumoral , Humanos , Interferência de RNA , Simplexvirus/fisiologiaRESUMO
The catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activity in vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activity in vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi's sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses. Importance: The herpesviruses cause considerable morbidity and mortality. Nucleoside analogs have served as effective antiviral agents against the herpesviruses, but resistance can arise through viral mutation. Second-line anti-herpes drugs have limitations in terms of pharmacokinetic properties and/or toxicity, so there is a great need for additional drugs for treatment of herpesviral infections. This study showed that the HIV integrase inhibitors also block herpesviral infection, raising the important potential of a new class of anti-herpes drugs and the prospect of drugs that combat both HIV and the herpesviruses.
Assuntos
Antivirais/farmacologia , Inibidores de Integrase de HIV/farmacologia , Simplexvirus/efeitos dos fármacos , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 8/efeitos dos fármacos , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
Like other DNA viruses that replicate in the nucleus, herpes simplex virus 1 (HSV-1) regulates the association of histones with its genome to promote viral replication and gene expression. We previously demonstrated that SNF2H, a member of the ISWI family of chromatin-remodeling factors, is concentrated in HSV-1 replication compartments in the nuclei of infected cells, suggesting that this cellular enzyme plays a role in viral replication. We show here that small interfering RNA (siRNA)-mediated knockdown of SNF2H in HEp-2 cells resulted in an approximately 20-fold decrease in HSV-1 replication, arguing that SNF2H promotes efficient HSV-1 replication. Decreases in HSV-1 replication were observed with multiple SNF2H-specific siRNAs, and the extent of the replication decrease correlated with the amount of SNF2H knockdown, indicating that the phenotype resulted from decreased SNF2H levels rather than off-target effects of the siRNAs. We also observed a decrease in the accumulation of immediate-early (IE) gene products in HSV-1-infected cells in which SNF2H was knocked down. Histone H3 occupancy on viral promoters was increased in HSV-1-infected cells that were transfected with SNF2H-specific siRNAs, suggesting that SNF2H promotes removal of histones from viral promoters during infection. Furthermore, chromatin immunoprecipitation (ChIP) studies showed that SNF2H associated with the HSV-1 genome during infection, which suggests that SNF2H may directly remodel viral chromatin. We hypothesize that SNF2H is recruited to viral promoters during HSV-1 infection, where it can remodel the chromatin state of the viral genome, facilitate the transcription of immediate-early genes, and enhance viral replication.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Viral da Expressão Gênica , Genes Precoces , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Replicação Viral , Adenosina Trifosfatases/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , HumanosRESUMO
The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal's activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal's ability to increase phosphorylation of eIF2 alpha during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells.
Assuntos
Antivirais/farmacologia , Cinamatos/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Tioureia/análogos & derivados , Proteínas Virais/fisiologia , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Expressão Gênica , Genes Virais , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Camundongos , Mutação , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Tioureia/farmacologia , Células Vero , Proteínas Virais/genéticaRESUMO
Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.