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Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.
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Human cytomegalovirus (HCMV) is the top viral cause of birth defects worldwide, and current therapies have high toxicity. We previously reported that the mTOR-independent autophagy-inducing disaccharide trehalose inhibits HCMV replication in multiple cell types. Here, we examine the mechanism of inhibition and introduce the autophagy inducer SMER28 as an additional inhibitor of HCMV acting through a different mechanism. We find that trehalose induces vacuolation and acidification of vacuoles and that debris, including debris with an appearance consistent with that of abnormal virions, is present in multivesicular bodies. Trehalose treatment increased the levels of Rab7, a protein required for lysosomal biogenesis and fusion, and slightly decreased the levels of Rab11, which is associated with recycling endosomes. We also present evidence that trehalose can promote autophagy without altering cellular glucose uptake. We show that SMER28 inhibits HCMV at the level of early protein production and interferes with viral genome replication in a cell type-dependent fashion. Finally, we show that SMER28 treatment does not cause the vacuolation, acidification, or redistribution of Rab7 associated with trehalose treatment and shows only a modest and cell type-dependent effect on autophagy. We propose a model in which the reciprocal effects on Rab7 and Rab11 induced by trehalose contribute to the redirection of enveloped virions from the plasma membrane to acidified compartments and subsequent degradation, and SMER28 treatment results in decreased expression levels of early and late proteins, reducing the number of virions produced without the widespread vacuolation characteristic of trehalose treatment.IMPORTANCE There is a need for less toxic HCMV antiviral drugs, and modulation of autophagy to control viral infection is a new strategy that takes advantage of virus dependence on autophagy inhibition. The present study extends our previous work on trehalose by showing a possible mechanism of action and introduces another autophagy-inducing compound, SMER28, that is effective against HCMV in several cell types. The mechanism by which trehalose induces autophagy is currently unknown, although our data show that trehalose does not inhibit cellular glucose uptake in cells relevant for HCMV replication but instead alters virion degradation by promoting acidic vacuolization. The comparison of our cell types and those used by others highlights the cell type-dependent nature of studying autophagy.
Assuntos
Compostos Alílicos/farmacologia , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/fisiologia , Quinazolinas/farmacologia , Trealose/farmacologia , Replicação Viral/efeitos dos fármacos , Autofagia/genética , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Humanos , Replicação Viral/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
UNLABELLED: Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem in immunocompromised individuals and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates but not in mammals and that induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease. IMPORTANCE: HCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the nontoxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of the development of resistance to treatment.
Assuntos
Autofagia/efeitos dos fármacos , Citomegalovirus/fisiologia , Trealose/metabolismo , Replicação Viral , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/virologiaRESUMO
UNLABELLED: Human cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C coactivator Cdh1 and by inducing the degradation of subunits APC4 and APC5, which along with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells, UL21a but not UL97 can disrupt APC/C function, leading to the accumulation of substrates. We find that UL21a is necessary and sufficient to induce the degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon the depletion of any one of these subunits or of subunit APC8. Finally, we show that at a low multiplicity of infection, either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by the expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using small interfering RNAs against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit downmodulation. IMPORTANCE: HCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in the control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform subcomplex of the APC/C, is an additional target of the degradation induced by HCMV protein UL21a. This study also shows for the first time that there is a unique cellular process in uninfected cells whereby depletion of APC1, APC4, APC5, or APC8 recapitulates the pattern of HCMV-mediated APC/C subunit degradation.
Assuntos
Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Citomegalovirus/fisiologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Proteínas Virais/metabolismo , Replicação Viral , Células Cultivadas , Interações Hospedeiro-Patógeno , HumanosRESUMO
UNLABELLED: Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development, as well as the state of differentiation of susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE: Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study, we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells, which are similar to those present in the developing neural tube, do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS.
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Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , DNA Viral/metabolismo , Células-Tronco Embrionárias/virologia , Células-Tronco Neurais/virologia , Diferenciação Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/fisiopatologia , DNA Viral/genética , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Células-Tronco Neurais/citologia , Replicação ViralRESUMO
Human cytomegalovirus (HCMV) infection modulates the host cell cycle to create an environment that is optimal for viral gene expression, DNA replication, and production of infectious virus. The virus mostly infects quiescent cells and thus must push the cell into G1 phase of the cell cycle to co-opt the cellular mechanisms that could be used for DNA synthesis. However, at the same time, cellular functions must be subverted such that synthesis of viral DNA is favored over that of the host. The molecular mechanisms by which this is accomplished include altered RNA transcription, changes in the levels and activity of cyclin-dependent kinases, and other proteins involved in cell cycle control, posttranslational modifications of proteins, modulation of protein stability through targeted effects on the ubiquitin-proteasome degradation pathway, and movement of proteins to different cellular locations. When the cell is in the optimal G0/G1 phase, multiple signaling pathways are altered to allow rapid induction of viral gene expression once negative factors have been eliminated. For the most part, the cell cycle will stop prior to initiation of host cell DNA synthesis (S phase), although many cell cycle proteins characteristic of the S/G2/M phase accumulate. The environment of a cell progressing through the cell cycle and dividing is not favorable for viral replication, and HCMV has evolved ways to sense whether cells are in S/G2 phase, and if so, to prevent initiation of viral gene expression until the cells cycle back to G1. A major target of HCMV is the anaphase-promoting complex E3 ubiquitin ligase, which is responsible for the ubiquitination and subsequent degradation of cyclins A and B and other cell cycle proteins at specific phases in the cell cycle. This review will discuss the effects of HCMV infection on cell cycle regulatory pathways, with the focus on selected viral proteins that are responsible for these effects.
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Ciclo Celular , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Pontos de Checagem do Ciclo Celular , Ciclina A/metabolismo , Infecções por Citomegalovirus/genética , Replicação do DNA , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.
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Hidroxicolesteróis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Ésteres do Colesterol/metabolismo , Perfilação da Expressão Gênica , Hidroxicolesteróis/metabolismo , Receptores X do Fígado , Macrófagos/citologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologia , Receptores Nucleares Órfãos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingolipídeos/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidoresRESUMO
Atherosclerosis is a major pathogenic factor in cardiovascular diseases, which are the leading cause of mortality in developed countries. While risk factors for atherosclerosis tend to be systemic, the distribution of atherosclerotic plaques within the vasculature is preferentially located at branch points and curves where blood flow is disturbed and shear stress is low. It is now widely accepted that hemodynamic factors can modulate endothelial gene expression and function and influence the pathophysiological changes associated with atherosclerosis. Human cytomegalovirus (HCMV), a ubiquitous pathogen, has long been proposed as a risk factor for atherosclerosis. To date, the role of HCMV in atherogenesis has been explored only in static conditions, and it is not known how HCMV infection is influenced by the physiological context of flow. In this study, we utilized a parallel-plate flow system to simulate the effects of shear stresses in different regions of the vasculature in vitro. We found that endothelial cells cultured under low shear stress, which simulates the flow condition of atheroprone regions in vivo, are more permissive to HCMV infection than cells experiencing high shear stress or static conditions. Cells exposed to low shear stress show increased entry of HCMV compared to cells exposed to high shear stress or static conditions. Viral structural gene expression, viral titers, and viral spread are also enhanced in endothelial cells exposed to low shear stress. These results suggest that hemodynamic factors modulate HCMV infection of endothelial cells, thus providing new insights into the induction/acceleration of atherosclerosis by HCMV.
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Artérias/virologia , Citomegalovirus/fisiologia , Endotélio Vascular/virologia , Fusão de Membrana , Sequência de Bases , Western Blotting , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Primers do DNA , DNA Viral/biossíntese , Imunofluorescência , Regulação Viral da Expressão Gênica , Genes Virais , Humanos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da PolimeraseRESUMO
Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to â¼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as. Both of these miRNAs were capable of functionally repressing synthetic targets in transient transfection experiments. Additionally, through cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago)-bound RNAs from infected cells, followed by high-throughput sequencing, we have obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Surprisingly, three HCMV miRNA precursors exhibited differential incorporation of their mature miRNA arms between Ago2 and Ago1 complexes. Host miRNA abundances were also affected by HCMV infection, with significant upregulation observed for an miRNA cluster containing miR-96, miR-182, and miR-183. In addition to miRNAs, we also identified novel forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection.
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Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Viral/genética , Sequência de Bases , Linhagem Celular , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , RNA Viral/metabolismo , Análise de Sequência de RNARESUMO
Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.
Assuntos
Capsídeo/metabolismo , Citomegalovirus/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Bases , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/genética , Humanos , Mutação , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Proteínas Virais/química , Replicação Viral/genética , NucleolinaRESUMO
The IE2 86 protein of human cytomegalovirus (HCMV) is essential for productive infection. The mutation of glutamine to arginine at position 548 of IE2 86 causes the virus to grow both slowly and to very low titers, making it difficult to study this mutant via infection. In this study, Q548R IE2 86 HCMV was produced on the complementing cell line 86F/40HA, which allowed faster and higher-titer production of mutant virus. The main defects observed in this mutant were greatly decreased expression of IE2 40, IE2 60, UL83, and UL84. Genome replication and the induction of cell cycle arrest were found to proceed at or near wild-type levels, and there was no defect in transitioning to early or late protein expression. Q548R IE2 86 was still able to interact with UL84. Furthermore, Q548R IE2 40 maintained the ability to enhance UL84 expression in a cotransfection assay. Microarray analysis of Q548R IE2 HCMV revealed that the US8, US9, and US29-32 transcripts were all significantly upregulated. These results further confirm the importance of IE2 in UL83 and UL84 expression as well as pointing to several previously unknown regions of the HCMV genome that may be regulated by IE2.
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Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Mutação de Sentido Incorreto , Transcrição Gênica , Proteínas Virais/biossíntese , Proteínas Virais/genética , Substituição de Aminoácidos/genética , Arginina/genética , Técnicas de Cultura de Células , Células Cultivadas , Citomegalovirus/patogenicidade , Perfilação da Expressão Gênica , Glutamina/genética , Humanos , Análise em Microsséries , Replicação ViralRESUMO
To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.
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Herpes Genital/prevenção & controle , Herpesvirus Humano 2/imunologia , Vacinas contra Herpesvirus/imunologia , Imunização Secundária/métodos , Vacinação/métodos , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , DNA Viral/genética , Feminino , Gânglios Espinais/virologia , Cobaias , Herpes Genital/imunologia , Herpes Genital/patologia , Herpesvirus Humano 2/genética , Vacinas contra Herpesvirus/administração & dosagem , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Prevenção Secundária , Vacinas de DNA/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
The emergence of Zika virus (ZIKV) as a global health threat has highlighted the unmet need for ZIKV-specific vaccines and antiviral treatments. ZIKV infects dendritic cells (DC), which have pivotal functions in activating innate and adaptive antiviral responses; however, the mechanisms by which DC function is subverted to establish ZIKV infection are unclear. Here we develop a genomics profiling method that enables discrete analysis of ZIKV-infected versus neighboring, uninfected primary human DCs to increase the sensitivity and specificity with which ZIKV-modulated pathways can be identified. The results show that ZIKV infection specifically increases the expression of genes enriched for lipid metabolism-related functions. ZIKV infection also increases the recruitment of sterol regulatory element-binding protein (SREBP) transcription factors to lipid gene promoters, while pharmacologic inhibition or genetic silencing of SREBP2 suppresses ZIKV infection of DCs. Our data thus identify SREBP2-activated transcription as a mechanism for promoting ZIKV infection amenable to therapeutic targeting.
Assuntos
Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Células Dendríticas , Humanos , Lipídeos , Transcrição GênicaRESUMO
We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus/genética , DNA Viral/biossíntese , Genes Virais , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Transcrição Gênica , Células Cultivadas , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Replicação do DNA , DNA Viral/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Fibroblastos/virologia , Hexosiltransferases , Humanos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma , Replicação ViralRESUMO
It has previously been demonstrated that, during human cytomegalovirus infection, the viral IE2 86 and IE2 40 proteins are both important for the expression of an early-late viral protein, UL84. Here, we show that expression of the UL84 protein is enhanced upon cotransfection with either IE2 86 or IE2 40, although IE2 40 appears to play a more important role. The UL84 protein levels are tightly linked to the amount of IE2 40 present, but this does not appear to be true for IE2 86. RNA remains constant for all corresponding proteins, indicating posttranscriptional regulation of UL84. The first 105 amino acids of UL84 are necessary and sufficient for this phenotype, and this region is also required for an interaction with IE2 86 and IE2 40. Treatment with proteasome inhibitors shows that UL84 exhibits some proteasome-dependent degradation, and UL84 is not protected against this degradation when coexpressed with IE2 86 or IE2 40. UL84 also exhibits an inhibitory effect on IE2 86 and IE2 40 protein levels in these cotransfection assays. Further, we show that the amino acid sequence of UL84 is important for the enhancement governed by IE2 40. These results indicate that IE2 86, IE2 40, and UL84 serve to regulate protein expression in a posttranscriptional fashion and that this regulation is independent of other viral proteins.
Assuntos
Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/fisiologia , Transativadores/fisiologia , Proteínas Virais/biossíntese , Sequência de Aminoácidos , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Proteínas Virais/genéticaRESUMO
Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G(1)/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.
Assuntos
Caderinas/metabolismo , Infecções por Citomegalovirus/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Antígenos CD , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Sequência de Bases , Sítios de Ligação/genética , Caderinas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Primers do DNA/genética , Geminina , Deleção de Genes , Genes Precoces , Genes Virais , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Inibidores de Proteassoma , Estabilidade Proteica , Subunidades Proteicas , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Human cytomegalovirus (HCMV) infection results in the formation of nuclear viral transcriptosomes, which are sites dedicated to viral immediate-early (IE) transcription. At IE times of the infection, viral and cellular factors, including several components of transcription such as cyclin-dependent kinase 9 (cdk9), localize at these sites. To determine the mechanism and requirements of specific recruitment of cdk9 to the viral transcriptosomes, infection in the presence of inhibitor drugs and infection of cell lines expressing exogenous mutant cdk9 were performed. We found that cdk9 localization to the viral transcriptosomes requires de novo protein synthesis. In addition, active transcription is required for recruitment and maintenance of cdk9 at the viral transcriptosomes. In cells infected with a recombinant IE2 HCMV (IE2 86 DeltaSX virus) in which IE2 gene expression is greatly reduced, cdk9 localization at the transcriptosome is delayed and corresponds to the kinetics of accumulation of the IE2 protein at these sites. Infection in the presence of the cdk9 inhibitors Flavopiridol and DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) allowed cdk9 localization to the viral transcriptosomes. A kinase-inactive cdk9 (D167N) expressed during the infection also localizes to the viral transcriptosomes, indicating that kinase activity of cdk9 is not a requirement for its localization to the sites of IE transcription. Exogenous expression of additional cdk9 mutants indicates that binding of Brd4 to the cdk9 complex is not required but that efficient binding to cyclin T1 is essential.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismo , Transcrição Gênica/genética , Proteínas de Ciclo Celular , Células Cultivadas , Ciclina T , Quinase 9 Dependente de Ciclina/genética , Ciclinas/genética , Citomegalovirus/genética , Infecções por Citomegalovirus , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Biossíntese de Proteínas/genética , RNA Viral/genética , Transativadores/genética , Fatores de Transcrição/metabolismoRESUMO
Infection with Herpes Simplex Viruses (HSVs) represents a significant health burden worldwide with HSV-1 and HSV-2 causing genital disease and HSV-2 contributing to human immunodeficiency virus acquisition. Despite great need, there is currently no licensed vaccine against HSV. In this report, we evaluated the protective efficacy of a vaccine containing highly purified, inactivated HSV-2 particles (with and without additional recombinant glycoprotein D) formulated with a monophosphoryl lipid A/Alhydrogel adjuvant in a guinea pig HSV genital model. The key results from 3 independent studies were: (1) vaccination consistently provided significant 3-3.5 Log10 reductions in vaginal HSV-2 titers on day 2 postchallenge; (2) following homologous or heterologous challenge with two U.S. isolates, all vaccine groups showed complete protection against lesion formation, significant 3 Log10 reductions in day 2 virus shedding, enhanced virus clearance, significant reductions in HSV-2 DNA within ganglia, and no detectable shedding (<2 PFU) or latent viral DNA in some immunized animals; (3) following challenge with a third heterologous strain, vaccination provided complete protection against primary and recurrent lesions, significant reductions in primary virus shedding, a 50% reduction in recurrent shedding days, and undetectable latent virus in the ganglia and spinal cords of most animals; and (4) adding glycoprotein D provided no enhanced protection relative to that elicited by the inactivated HSV-2 particles alone. Together, these data provide strong support for further development of this exceedingly protective and highly feasible vaccine candidate for human trials.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Herpes Genital/prevenção & controle , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Herpesvirus Humano 2/efeitos dos fármacos , Vírion , Administração Intravaginal , Animais , Chlorocebus aethiops , Feminino , Cobaias , Herpes Genital/imunologia , Vacinas contra o Vírus do Herpes Simples/imunologia , Herpesvirus Humano 2/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Células Vero , Vírion/imunologia , Eliminação de Partículas Virais/efeitos dos fármacos , Eliminação de Partículas Virais/imunologiaRESUMO
We previously reported that defined components of the host transcription machinery are recruited to human cytomegalovirus immediate-early (IE) transcription sites, including cdk9 and cdk7 (S. Tamrakar, A. J. Kapasi, and D. H. Spector, J. Virol. 79:15477-15493, 2005). In this report, we further document the complexity of this site, referred to as the transcriptosome, through identification of additional resident proteins, including viral UL69 and cellular cyclin T1, Brd4, histone deacetylase 1 (HDAC1), and HDAC2. To examine the role of cyclin-dependent kinases (cdks) in the establishment of this site, we used roscovitine, a specific inhibitor of cdk1, cdk2, cdk7, and cdk9, that alters processing of viral IE transcripts and inhibits expression of viral early genes. In the presence of roscovitine, IE2, cyclin T1, Brd4, HDAC1, and HDAC2 accumulate at the transcriptosome. However, accumulation of cdk9 and cdk7 was specifically inhibited. Roscovitine treatment also resulted in decreased levels of cdk9 and cdk7 RNA. There was a corresponding reduction in cdk9 protein but only a modest decrease in cdk7 protein. However, overexpression of cdk9 does not compensate for the effects of roscovitine on cdk9 localization or viral gene expression. Delaying the addition of roscovitine until 8 h postinfection prevented all of the observed effects of the cdk inhibitor. These data suggest that IE2 and multiple cellular factors needed for viral RNA synthesis accumulate within the first 8 h at the viral transcriptosome and that functional cdk activity is required for the specific recruitment of cdk7 and cdk9 during this time interval.