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1.
J Virol ; 96(11): e0039822, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35543552

RESUMO

Poxvirus proteins remodel signaling throughout the cell by targeting host enzymes for inhibition and redirection. Recently, it was discovered that early in infection the vaccinia virus (VACV) B12 pseudokinase copurifies with the cellular kinase VRK1, a proviral factor, in the nucleus. Although the formation of this complex correlates with inhibition of cytoplasmic VACV DNA replication and likely has other downstream signaling consequences, the molecular mechanisms involved are poorly understood. Here, we further characterize how B12 and VRK1 regulate one another during poxvirus infection. First, we demonstrate that B12 is stabilized in the presence of VRK1 and that VRK1 and B12 coinfluence their respective solubility and subcellular localization. In this regard, we find that B12 promotes VRK1 colocalization with cellular DNA during mitosis and that B12 and VRK1 may be tethered cooperatively to chromatin. Next, we observe that the C-terminal tail of VRK1 is unnecessary for B12-VRK1 complex formation or its proviral activity. Interestingly, we identify a point mutation of B12 capable of abrogating interaction with VRK1 and which renders B12 nonrepressive during infection. Lastly, we investigated the influence of B12 on the host factor BAF and antiviral signaling pathways and find that B12 triggers redistribution of BAF from the cytoplasm to the nucleus. In addition, B12 increases DNA-induced innate immune signaling, revealing a new functional consequence of the B12 pseudokinase. Together, this study characterizes the multifaceted roles B12 plays during poxvirus infection that impact VRK1, BAF, and innate immune signaling. IMPORTANCE Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction. While the multifaceted roles pseudokinases can play are known, their contribution to virus infection remains understudied. Here, we further characterize the mechanism of how the VACV B12 pseudokinase and human VRK1 kinase regulate one another in the nucleus during poxvirus infection and inhibit VACV DNA replication. We find that B12 disrupts regulation of VRK1 and its downstream target BAF, while also enhancing DNA-dependent innate immune signaling. Combined with previous data, these studies contribute to the growing field of nuclear pathways targeted by poxviruses and provide evidence of unexplored roles of B12 in the activation of antiviral immunity.


Assuntos
Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular , Infecções por Poxviridae , Proteínas Serina-Treonina Quinases , Vaccinia virus , DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Infecções por Poxviridae/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Vacínia , Vaccinia virus/enzimologia , Vaccinia virus/fisiologia
2.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33177193

RESUMO

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulates a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia-related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phosphoinactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia virus infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms that poxviruses use to hijack homologous cellular signaling pathways during infection.IMPORTANCE Viruses from diverse families encode both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Here, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vaccinia virus/enzimologia , Vacínia/imunologia , Proteínas Virais/metabolismo , Antivirais , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Vacínia/metabolismo , Vacínia/virologia , Proteínas Virais/genética
3.
PLoS Pathog ; 15(2): e1007608, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30768651

RESUMO

Poxviruses employ sophisticated, but incompletely understood, signaling pathways that engage cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. For example, the vaccinia B1 protein kinase plays a vital role in inactivating the cellular antiviral factor BAF, and likely orchestrates other pathways as well. In this study, we utilized experimental evolution of a B1 deletion virus to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. After several passages of the ΔB1 virus we observed a robust increase in viral titer of the adapted virus. Interestingly, our characterization of the adapted viruses reveals that mutations correlating with a loss of function of the vaccinia B12 pseudokinase provide a striking fitness enhancement to this virus. In support of predictions that reductive evolution is a driver of poxvirus adaptation, this is clear experimental evidence that gene loss can be of significant benefit. Next, we present multiple lines of evidence demonstrating that expression of full length B12 leads to a fitness reduction in viruses with a defect in B1, but has no apparent impact on wild-type virus or other mutant poxviruses. From these data we infer that B12 possesses a potent inhibitory activity that can be masked by the presence of the B1 kinase. Further investigation of B12 attributes revealed that it primarily localizes to the nucleus, a characteristic only rarely found among poxviral proteins. Surprisingly, BAF phosphorylation is reduced under conditions in which B12 is present in infected cells without B1, indicating that B12 may function in part by enhancing antiviral activity of BAF. Together, our studies of B1 and B12 present novel evidence that a paralogous kinase-pseudokinase pair can exhibit a unique epistatic relationship in a virus, perhaps serving to enhance B1 conservation during poxvirus evolution and to orchestrate yet-to-be-discovered nuclear events during infection.


Assuntos
Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Células A549 , Animais , Linhagem Celular , Núcleo Celular , Chlorocebus aethiops , Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Proteínas Nucleares , Fosforilação , Poxviridae/metabolismo , Poxviridae/patogenicidade , Proteínas Quinases , Transdução de Sinais , Fatores de Transcrição , Vacínia , Vaccinia virus/patogenicidade , Proteínas Virais/genética , Proteínas Virais/fisiologia , Replicação Viral/genética
4.
J Virol ; 93(20)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31341052

RESUMO

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus's dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner.IMPORTANCE Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals, including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia virus B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia virus B12 protein via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.


Assuntos
Interações Hospedeiro-Patógeno , Proteínas Serina-Treonina Quinases/metabolismo , Vaccinia virus/fisiologia , Vacínia/metabolismo , Vacínia/virologia , Replicação Viral , Linhagem Celular , Células Cultivadas , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Mutação , Fosforilação , Vaccinia virus/classificação
5.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28515294

RESUMO

The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.


Assuntos
Deleção de Genes , Interações Hospedeiro-Patógeno , Proteínas Serina-Treonina Quinases/metabolismo , Vaccinia virus/enzimologia , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Replicação do DNA , Humanos , Proteínas Virais/genética
6.
J Virol ; 90(8): 3806-3809, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26842478

RESUMO

Intrinsic defenses targeting foreign DNA are one facet of the cellular armament tasked with protecting host genomic integrity. The DNA binding protein BAF (barrier to autointegration factor) contributes to multiple aspects of genome maintenance and intercepts retrovirus, poxvirus, and herpesvirus genomes during infection. In this gem, we discuss the unique position BAF occupies at the virus-host interface and how both viral and cellular mechanisms may regulate its capacity to act as a pro- or antiviral effector targeting viral DNA.


Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Proteínas Nucleares/metabolismo , Integração Viral , Humanos
7.
J Virol ; 89(20): 10247-59, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26223647

RESUMO

UNLABELLED: The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive temperature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at another stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demonstrated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell type-specific regulation of BAF and reveals a role for B1 beyond DNA replication during the late stages of the viral life cycle. IMPORTANCE: The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been postulated for decades but are difficult to examine directly due to the importance of B1 during DNA replication. Here, we demonstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both dependent on and independent of BAF.


Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Osteoblastos/virologia , Proteínas Quinases/genética , Vaccinia virus/genética , Proteínas Virais/genética , Animais , Fusão Celular , Linhagem Celular Tumoral , DNA Viral/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Osteoblastos/imunologia , Proteínas Quinases/imunologia , Transdução de Sinais , Vaccinia virus/imunologia , Proteínas Virais/imunologia , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/imunologia , Replicação Viral/genética
8.
J Virol ; 88(10): 5342-55, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24600006

RESUMO

UNLABELLED: Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. IMPORTANCE: The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Vaccinia virus/imunologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/química , Chlorocebus aethiops , Citosol/química , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica
9.
J Cell Sci ; 124(Pt 15): 2654-65, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21750191

RESUMO

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
10.
J Virol ; 86(11): 6197-209, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22438556

RESUMO

Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ∼200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vaccinia virus/enzimologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Citoplasma/química , Análise Mutacional de DNA , DNA Viral/metabolismo , Inativação Gênica , Humanos , Viabilidade Microbiana , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Vaccinia virus/genética
11.
J Virol ; 85(22): 11588-600, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21880762

RESUMO

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.


Assuntos
Proteínas de Ligação a DNA/imunologia , Interações Hospedeiro-Patógeno , Proteínas Nucleares/imunologia , Vaccinia virus/imunologia , Integração Viral/imunologia , Animais , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica
12.
Viruses ; 14(2)2022 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-35216024

RESUMO

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants, we characterise the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1 kinase and/or B12 pseudokinase mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins. (209/200.).


Assuntos
Ciclo Celular/fisiologia , Dano ao DNA , Interações Hospedeiro-Patógeno/genética , Vaccinia virus/genética , Proteínas Virais/genética , Proliferação de Células , Células HCT116 , Células HeLa , Humanos , Mutação , Proteína Supressora de Tumor p53 , Vaccinia virus/fisiologia , Replicação Viral
13.
Biol Reprod ; 82(1): 182-93, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19696012

RESUMO

The VRK1 protein kinase has been implicated as a pro-proliferative factor. Genetic analyses of mutant alleles of the Drosophila and Caenorhabditis elegans VRK1 homologs have revealed phenotypes ranging from embryonic lethality to mitotic and meiotic defects with resultant sterility. Herein, we describe the first genetic analysis of murine VRK1. Two lines of mice containing distinct gene-trap integrations into the Vrk1 locus were established. Insertion into intron 12 (GT12) spared VRK1 function, enabling the examination of VRK1 expression in situ. Insertion into intron 3 (GT3) disrupted VRK1 function, but incomplete splicing to the gene trap rendered this allele hypomorphic (approximately 15% of wild-type levels of VRK1 remain). GT3/GT3 mice are viable, but both males and females are infertile. In testes, VRK1 is expressed in Sertoli cells and spermatogonia. The infertility of GT3/GT3 male mice results from a progressive defect in spermatogonial proliferation or differentiation, culminating in the absence of mitotic and meiotic cells in adult testis. These data demonstrate an important role for VRK1 in cell proliferation and confirm that the need for VRK1 during gametogenesis is evolutionarily conserved.


Assuntos
Proliferação de Células , Infertilidade Masculina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células de Sertoli/metabolismo , Espermatogônias/metabolismo , Alelos , Animais , Antígenos Nucleares/metabolismo , Feminino , Infertilidade Feminina/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Serina-Treonina Quinases/genética
14.
Mol Biol Cell ; 17(5): 2451-64, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16495336

RESUMO

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N' terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/enzimologia , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoaminoácidos/análise , Fosforilação , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Serina/metabolismo , Treonina/metabolismo
15.
Methods Mol Biol ; 2023: 93-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31240672

RESUMO

This protocol describes how to couple two techniques, the generation of complementing cells lines and production of viral deletion mutants, to rapidly construct novel tools for poxvirus analysis. Specifically, the production and utilization of a complementing cell line expressing a poxvirus gene of interest are critical for the generation of poxvirus mutants in which essential genes are disrupted. Complementing cells are also valuable for the characterization of vaccinia genes in the absence of infection. Here, we detail the process of isolating vaccinia virus deletion mutants. Deletion mutant generation involves homologous recombination between replicating viral DNA and transfected DNA followed by selection and screening on a complementing cell line that provides the deleted gene in trans. Finally, deletion is confirmed by polymerase chain reaction, sequencing, and functional assays if available.


Assuntos
DNA Viral/genética , Vaccinia virus/genética , Proteínas Virais/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , Deleção de Genes , Deleção de Sequência/genética , Deleção de Sequência/fisiologia , Proteínas Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
16.
Gene Expr ; 12(3): 193-212, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16128003

RESUMO

NF-Y is a bifunctional transcription factor capable of activating or repressing transcription. NF-Y specifically recognizes CCAAT box motifs present in many eukaryotic promoters. The mechanisms involved in regulating its activity are poorly understood. Previous studies have shown that the FGF-4 promoter is regulated positively by its CCAAT box and NF-Y in embryonal carcinoma (EC) cells where the distal enhancer of the FGF-4 gene is active. Here, we demonstrate that the CCAAT box functions as a negative cis-regulatory element when cis-regulatory elements of the FGF-4 enhancer are disrupted, or after EC cells differentiate and the FGF-4 enhancer is inactivated. We also demonstrate that NF-Y mediates the repression of the CCAAT box and that NF-Y associates with the endogenous FGF-4 gene in both EC cells and EC-differentiated cells. Importantly, we also determined that the orientation and the position of the CCAAT box are critical for its role in regulating the FGF-4 promoter. Together, these studies demonstrate that the distal enhancer of the FGF-4 gene determines whether the CCAAT box of the FGF-4 promoter functions as a positive or a negative cis-regulatory element. In addition, these studies are consistent with NF-Y playing an architectural role in its regulation of the FGF-4 promoter.


Assuntos
Fator de Ligação a CCAAT/fisiologia , Elementos Facilitadores Genéticos , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Carcinoma Embrionário/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Genes Reporter , Imunoprecipitação , Camundongos , Modelos Genéticos , Plasmídeos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção
17.
Curr Opin Cell Biol ; 34: 61-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26072104

RESUMO

The Barrier to Autointegration Factor (BAF or BANF1) is an abundant, highly conserved DNA binding protein. BAF is involved in multiple pathways including mitosis, nuclear assembly, viral infection, chromatin and gene regulation and the DNA damage response. BAF is also essential for early development in metazoans and relevant to human physiology; BANF1 mutations cause a progeroid syndrome, placing BAF within the laminopathy disease spectrum. This review summarizes previous knowledge about BAF in the context of recent discoveries about its protein partners, posttranslational regulation, dynamic subcellular localizations and roles in disease, innate immunity, transposable elements and genome integrity.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma , Imunidade Inata , Proteínas Nucleares/metabolismo , Progéria/genética , Progéria/metabolismo , Estresse Fisiológico , Animais , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/genética
18.
PLoS One ; 9(6): e100511, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24945635

RESUMO

BAF (Barrier to Autointegration Factor) is a highly conserved DNA binding protein that senses poxviral DNA in the cytoplasm and tightly binds to the viral genome to interfere with DNA replication and transcription. To counteract BAF, a poxviral-encoded protein kinase phosphorylates BAF, which renders BAF unable to bind DNA and allows efficient viral replication to occur. Herein, we examined how BAF phosphorylation is affected by herpes simplex virus type 1 (HSV-1) infection and tested the ability of BAF to interfere with HSV-1 productive infection. Interestingly, we found that BAF phosphorylation decreases markedly following HSV-1 infection. To determine whether dephosphorylated BAF impacts HSV-1 productive infection, we employed cell lines stably expressing a constitutively unphosphorylated form of BAF (BAF-MAAAQ) and cells overexpressing wild type (wt) BAF for comparison. Although HSV-1 production in cells overexpressing wtBAF was similar to that in cells expressing no additional BAF, viral growth was reduced approximately 80% in the presence of BAF-MAAAQ. Experiments were also performed to determine the mechanism of the antiviral activity of BAF with the following results. BAF-MAAAQ was localized to the nucleus, whereas wtBAF was dispersed throughout cells prior to infection. Following infection, wtBAF becomes dephosphorylated and relocalized to the nucleus. Additionally, BAF was associated with the HSV-1 genome during infection, with BAF-MAAAQ associated to a greater extent than wtBAF. Importantly, unphosphorylated BAF inhibited both viral DNA replication and gene expression. For example, expression of two regulatory proteins, ICP0 and VP16, were substantially reduced in cells expressing BAF-MAAAQ. However, other viral genes were not dramatically affected suggesting that expression of certain viral genes can be differentially regulated by unphosphorylated BAF. Collectively, these results suggest that BAF can act in a phosphorylation-regulated manner to impair HSV-1 transcription and/or DNA replication, which is similar to the antiviral activity of BAF during vaccinia infection.


Assuntos
Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/química , Herpes Simples/genética , Herpesvirus Humano 1/genética , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Fosforilação , Ligação Proteica , Transporte Proteico , Frações Subcelulares/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
19.
Virology ; 444(1-2): 363-73, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23891157

RESUMO

Barrier to autointegration factor (BAF/BANF1) is a cellular DNA-binding protein found in the nucleus and cytoplasm. Cytoplasmic BAF binds to foreign DNA and can act as a defense against vaccinia DNA replication. To evade BAF, vaccinia expresses the B1 kinase, which phosphorylates BAF and blocks its ability to bind DNA. Interestingly, B1 is also needed for viral intermediate gene expression via an unknown mechanism. Therefore, we evaluated the impact of B1-BAF signaling on vaccinia transcription. Strikingly, the decrease in vaccinia transcription caused by loss of B1 can be rescued by depletion of BAF. The repressive action of BAF is greatest on a viral promoter, and is more modest when non-vaccinia promoters are employed, which suggests BAF acts in a gene specific manner. These studies expand our understanding of the role of the B1 kinase during infection and provide the first evidence that BAF is a defense against viral gene expression.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Proteínas Nucleares/antagonistas & inibidores , Transcrição Gênica , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Proteínas de Ligação a DNA/imunologia , Humanos , Proteínas Nucleares/imunologia , Vaccinia virus/imunologia
20.
Cell Host Microbe ; 1(3): 187-97, 2007 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-18005698

RESUMO

Barrier to autointegration factor (BAF) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. Herein, we demonstrate a cytoplasmic role for BAF in host defense during poxviral infections. Vaccinia is the prototypic poxvirus, a family of DNA viruses that replicate exclusively in the cytoplasm of infected cells. Mutations in the vaccinia B1 kinase (B1) compromise viral DNA replication, but the mechanism by which B1 achieves this has remained elusive. We now show that BAF acts as a potent inhibitor of poxvirus replication unless its DNA-binding activity is blocked by B1-mediated phosphorylation. These data position BAF as the effector of an innate immune response that prevents replication of exogenous viral DNA in the cytoplasm. To enable the virus to evade this defense, the poxviral B1 has evolved to usurp a signaling pathway employed by the host cell.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Interações Hospedeiro-Patógeno/imunologia , Proteínas Nucleares/antagonistas & inibidores , Vaccinia virus/enzimologia , Vaccinia virus/imunologia , Proteínas Virais/metabolismo , Replicação Viral/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , DNA Viral/biossíntese , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Virais/genética , Replicação Viral/fisiologia
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