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1.
PLoS Pathog ; 20(1): e1011907, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38232124

RESUMO

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.


Assuntos
Herpesvirus Humano 8 , Proteínas Nucleares , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiologia , Latência Viral/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Expressão Gênica , Regulação Viral da Expressão Gênica , Replicação Viral
2.
Nucleic Acids Res ; 52(4): 1814-1829, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38180827

RESUMO

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/genética , Latência Viral/genética , DNA , Reparo do DNA
3.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34725152

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the endothelial tumor KS, a leading cause of morbidity and mortality in sub-Saharan Africa. KSHV-encoded microRNAs (miRNAs) are known to play an important role in viral oncogenesis; however, the role of host miRNAs in KS tumorigenesis remains largely unknown. Here, high-throughput small-RNA sequencing of the cellular transcriptome in a KS xenograft model revealed miR-127-3p as one of the most significantly down-regulated miRNAs, which we validated in KS patient tissues. We show that restoration of miR-127-3p suppresses KSHV-driven cellular transformation and proliferation and induces G1 cell cycle arrest by directly targeting the oncogene SKP2. This miR-127-3p-induced G1 arrest is rescued by disrupting the miR-127-3p target site in SKP2 messenger RNA (mRNA) using gene editing. Mechanistically, miR-127-3p-mediated SKP2 repression elevates cyclin-dependent kinase (CDK) inhibitor p21Cip1 and down-regulates cyclin E, cyclin A, and CDK2, leading to activation of the RB protein tumor suppressor pathway and suppression of the transcriptional activities of E2F and Myc, key oncoprotein transcription factors crucial for KSHV tumorigenesis. Consequently, metabolomics analysis during miR-127-3p-induced cell cycle arrest revealed significant depletion of dNTP pools, consistent with RB-mediated repression of key dNTP biosynthesis enzymes. Furthermore, miR-127-3p reconstitution in a KS xenograft mouse model suppresses KSHV-positive tumor growth by targeting SKP2 in vivo. These findings identify a previously unrecognized tumor suppressor function for miR-127-3p in KS and demonstrate that the miR-127-3p/SKP2 axis is a viable therapeutic strategy for KS.


Assuntos
Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Sarcoma de Kaposi/metabolismo , Animais , Carcinogênese , Feminino , Herpesvirus Humano 8/fisiologia , Humanos , Camundongos Nus , Sarcoma de Kaposi/virologia
4.
Nucleic Acids Res ; 49(22): 12895-12911, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34850113

RESUMO

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.


Assuntos
Antígenos Virais/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Sarcoma de Kaposi/genética , Latência Viral/genética , Antígenos Virais/química , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/fisiologia , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Sarcoma de Kaposi/virologia
5.
Proc Natl Acad Sci U S A ; 117(36): 22443-22451, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820070

RESUMO

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA's acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.


Assuntos
Antígenos Virais/genética , Antígenos Virais/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Acetilação , Animais , Antígenos Virais/química , DNA Viral/genética , Feminino , Células HEK293 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/química , Plasmídeos/genética
6.
PLoS Pathog ; 15(2): e1007628, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30811506

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects host cells and establishes lifelong persistence as an extra-chromosomal episome in the nucleus. To persist in proliferating cells, the viral genome typically replicates once per cell cycle and is distributed into daughter cells. This process involves host machinery utilized by KSHV, however the underlying mechanisms are not fully elucidated. In present study, we found that N-Myc downstream regulated gene 1 (NDRG1), a cellular gene known to be non-detectable in primary B cells and endothelial cells which are the major cell types for KSHV infection in vivo, was highly upregulated by KSHV in these cells. We further demonstrated that the high expression of NDRG1 was regulated by latency-associated nuclear antigen (LANA), the major viral latent protein which tethers the viral genome to host chromosome and plays an essential role in viral genome maintenance. Surprisingly, knockdown of NDRG1 in KSHV latently infected cells resulted in a significant decrease of viral genome copy number in these cells. Interestingly, NDRG1 can directly interact with proliferating cell nuclear antigen (PCNA), a cellular protein which functions as a DNA polymerase clamp during DNA replication. Intriguingly, we found that NDRG1 forms a complex with LANA and PCNA and serves as a scaffold protein bridging these two proteins. We further demonstrated that NDRG1 is critical for mediating LANA to recruit PCNA onto terminal repeat (TR) of KSHV genome, and facilitates viral DNA replication and episome persistence. Taken together, our findings suggest that NDRG1 plays an important role in KSHV viral genome replication, and provide new clues for understanding of KSHV persistence.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Adulto , Antígenos Virais/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Replicação do DNA , DNA Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Viral , Células HEK293 , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Regulação para Cima , Latência Viral , Replicação Viral
7.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626680

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.


Assuntos
Antígenos Virais/genética , Herpesvirus Humano 8/genética , Proteínas Nucleares/genética , Plasmídeos/genética , Sarcoma de Kaposi/virologia , Antígenos Nucleares/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/virologia , Cromossomos/genética , Replicação do DNA/genética , DNA Viral/genética , Genoma Viral/genética , Células HEK293 , Humanos , Mitose/genética , Sequências Repetidas Terminais/genética , Proteínas Virais/genética , Latência Viral/genética , Replicação Viral/genética
8.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30111565

RESUMO

The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4.IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.


Assuntos
Antígenos Virais/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 8/genética , Proteínas Nucleares/metabolismo , Rhadinovirus/genética , Sequências Repetidas Terminais/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , DNA Viral/genética , Genoma Viral/genética , Camundongos , Latência Viral/genética
9.
PLoS Pathog ; 13(9): e1006555, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28910389

RESUMO

Many pathogens, including Kaposi's sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.


Assuntos
Antígenos Virais/metabolismo , DNA Viral/genética , Genoma Viral/genética , Centro Germinativo/metabolismo , Herpesvirus Humano 8 , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Sarcoma de Kaposi/metabolismo , Latência Viral/genética , Animais , Antígenos Virais/genética , Linfócitos B/metabolismo , Linfócitos B/virologia , Camundongos , Proteínas Nucleares/genética , Plasmídeos/genética , Sarcoma de Kaposi/virologia
12.
Proc Natl Acad Sci U S A ; 113(49): 14121-14126, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27864512

RESUMO

Epstein-Barr virus (EBV) super-enhancers (ESEs) are essential for lymphoblastoid cell (LCL) growth and survival. Reanalyses of LCL global run-on sequencing (Gro-seq) data found abundant enhancer RNAs (eRNAs) being transcribed at ESEs. Inactivation of ESE components, EBV nuclear antigen 2 (EBNA2) and bromodomain-containing protein 4 (BRD4), significantly decreased eRNAs at ESEs -428 and -525 kb upstream of the MYC oncogene transcription start site (TSS). shRNA knockdown of the MYC -428 and -525 ESE eRNA caused LCL growth arrest and reduced cell growth. Furthermore, MYC ESE eRNA knockdown also significantly reduced MYC expression, ESE H3K27ac signals, and MYC ESEs looping to MYC TSS. These data indicate that ESE eRNAs strongly affect cell gene expression and enable LCL growth.


Assuntos
Elementos Facilitadores Genéticos , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Transtornos Linfoproliferativos/virologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Endonucleases , Humanos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
13.
Proc Natl Acad Sci U S A ; 112(32): E4354-63, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26195743

RESUMO

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas e Procedimentos Diagnósticos/instrumentação , Eletricidade , Nanoestruturas/química , Linhagem Celular Tumoral , Coinfecção/diagnóstico , Meio Ambiente , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Microfluídica , Concentração Osmolar , Reprodutibilidade dos Testes , Temperatura
14.
J Virol ; 90(17): 7667-83, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27307564

RESUMO

UNLABELLED: Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5S(mLANA)) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease. IMPORTANCE: The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure, and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.


Assuntos
Antígenos Virais/metabolismo , Linfócitos B/fisiologia , Proliferação de Células , Centro Germinativo/citologia , Interações Hospedeiro-Patógeno , Proteínas Nucleares/metabolismo , Rhadinovirus/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antígenos Virais/genética , Análise Mutacional de DNA , DNA Viral/metabolismo , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Ligação Proteica
15.
J Immunol ; 194(6): 2746-56, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25662997

RESUMO

CD4(+) T cells are critical for the control of virus infections, T cell memory, and immune surveillance. We studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4(+) T cells using gp150-specific TCR-transgenic mice. This allowed a more detailed study of the characteristics of the CD4(+) T cell response than did previously available approaches for this virus. Most gp150-specific CD4(+) T cells expressed T-bet and produced IFN-γ, indicating that MHV-68 infection triggered differentiation of CD4(+) T cells largely into the Th1 subset, whereas some became follicular Th cells and Foxp3(+) regulatory T cells. These CD4(+) T cells were protective against MHV-68 infection in the absence of CD8(+) T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4(+) T cells, based on lymphocyte Ag 6C (Ly6C) expression. Ly6C expression positively correlated with IFN-γ, TNF-α, and granzyme B production; T-bet and KLRG1 expression; proliferation; and CD4(+) T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression, and secondary expansion potential. Ly6C(+) and Ly6C(-) gp150-specific CD4(+) T cells were able to interconvert in a bidirectional manner upon secondary Ag exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4(+) T cells but is inversely correlated with memory potential. Interconversion between Ly6C(+) and Ly6C(-) cells may maintain a balance between the two Ag-specific CD4(+) T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4(+) T cells during persistent virus infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por Herpesviridae/imunologia , Rhadinovirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Apoptose/genética , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citometria de Fluxo , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Rhadinovirus/fisiologia , Baço/imunologia , Baço/metabolismo , Baço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia
16.
Nucleic Acids Res ; 43(20): 10039-54, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26424851

RESUMO

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.


Assuntos
Antígenos Virais/química , DNA Viral/química , Herpesvirus Humano 8 , Proteínas Nucleares/química , Rhadinovirus , Antígenos Virais/genética , Antígenos Virais/metabolismo , Sítios de Ligação , DNA Viral/metabolismo , Modelos Moleculares , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Sequências Repetidas Terminais , Termodinâmica
17.
J Biol Chem ; 290(47): 28084-28096, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26420481

RESUMO

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.


Assuntos
Antígenos Virais/metabolismo , Replicação do DNA , Herpesvirus Humano 8/genética , Proteínas Nucleares/metabolismo , Plasmídeos/fisiologia , Latência Viral , Sítios de Ligação , Linhagem Celular Tumoral , Herpesvirus Humano 8/imunologia , Humanos , Eletricidade Estática , Sequências Repetidas Terminais
18.
PLoS Pathog ; 9(8): e1003554, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23950719

RESUMO

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5S(mLANA) modulates cellular control of Myc turnover by antagonizing SCF(Fbw7) mediated proteasomal degradation of Myc, mimicking SCF(ß-TrCP). The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.


Assuntos
Infecções por Herpesviridae/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Rhadinovirus/metabolismo , Infecções Tumorais por Vírus/metabolismo , Ubiquitinação , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Humanos , Camundongos , Camundongos Knockout , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Rhadinovirus/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética
19.
PLoS Pathog ; 9(10): e1003673, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146618

RESUMO

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Šresolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).


Assuntos
DNA Viral/química , Dobramento de Proteína , Rhadinovirus/química , Proteínas Virais/química , Latência Viral , DNA Viral/genética , DNA Viral/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Rhadinovirus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
J Virol ; 87(22): 12270-83, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24006437

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.


Assuntos
Antígenos Virais/metabolismo , Replicação do DNA/genética , Herpesvirus Humano 8/fisiologia , Mutação/genética , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Sequências Repetidas Terminais/genética , Antígenos Virais/genética , Western Blotting , Núcleo Celular/genética , DNA Viral/genética , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Genoma Viral , Meia-Vida , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/virologia , Microscopia de Fluorescência , Proteínas Nucleares/genética , Ligação Proteica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Células Tumorais Cultivadas
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