KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

doi:10.1038/srep32633

Cited by 15 publications

(10 citation statements)

References 70 publications

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“…As well as these previously characterized NS5A partners, we also focused on the nucleosome assembly protein 1-like proteins 1 and 4 (NAP1L1 and NAP1L4). NAP1L1 has been shown to interact with HCV core and NS5A ( 33 , 34 ) as well as Kaposi's sarcoma herpesvirus (KSHV) LANA ( 35 ) and HIV-1 Tat ( 36 ). Although its roles in the nucleus associated with chromatin structure and gene expression are well defined, NAP1L1 is predominantly located in the cytoplasm, where its function(s) remains to be elucidated.…”

Section: Resultsmentioning

confidence: 99%

“…Of the five mammalian NAP1 family members (NAP1L1 to NAP1L5), only NAP1L1 and NAP1L4 are ubiquitously expressed, while the other three are expressed mainly in neurons ( 41 ). NAPs have been shown to interact with proteins of a number of DNA viruses, including papillomavirus E2 ( 42 ), Epstein-Barr virus EBNA1 ( 43 ), and KSHV LANA ( 35 ). NAP1L1 also binds to HIV-1 Tat and Rev, and these interactions regulate viral infectivity and the subcellular localization of the virus proteins ( 36 , 44 ).…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Goonawardane

Gebhardt

Bartlett

et al. 2017

J Virol

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Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Goonawardane

Gebhardt

Bartlett

et al. 2017

J Virol

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“…This observation suggests that silencing of gene expression via recruitment of DNA methyltransferases is not the universal outcome of LANA binding but instead may be restricted to a few host genes such as TβRII and CDH13 [120,121], perhaps in a contextspecific manner (of note, these two sites were not recovered in the three ChIP-seq studies, but this may be due to the higher sensitivity of the specific PCR that was used in the previous studies). While LANA binding therefore seems to be indolent LANA recruits NAP1L1 to viral terminal repeats to regulate nucleosome assembly and gene expression [116] PEL cell lines (BCBL-1, BC-3); ectopic expression (HEK293T, HEK293L) LANA interferes with the interaction between CIITA and RFX components, resulting in reduced MHC-II promoter activity [117] PEL cell lines (BCBL-1, BC-3); ectopic expression (HEK293T, HEK293L, BJAB, THP-1, and DG75)…”

Section: Viral Manipulation Of the Host Epigenomementioning

confidence: 99%

Epigenetic control in Kaposi sarcoma-associated herpesvirus infection and associated disease

Fröhlich

Grundhoff

2020

Semin Immunopathol

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Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several malignancies of endothelial and B-cell origin. The fact that latently infected tumor cells in these malignancies do not express classical viral oncogenes suggests that pathogenesis of KSHV-associated disease results from multistep processes that, in addition to constitutive viral gene expression, may require accumulation of cellular alterations. Heritable changes of the epigenome have emerged as an important co-factor that contributes to the pathogenesis of many non-viral cancers. Since KSHV encodes a number of factors that directly or indirectly manipulate host cell chromatin, it is an intriguing possibility that epigenetic reprogramming also contributes to the pathogenesis of KSHV-associated tumors. The fact that heritable histone modifications have also been shown to regulate viral gene expression programs in KSHV-infected tumor cells underlines the importance of epigenetic control during latency and tumorigenesis. We here review what is presently known about the role of epigenetic regulation of viral and host chromatin in KSHV infection and discuss how viral manipulation of these processes may contribute to the development of KSHV-associated disease.

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“…transducing an shLANA lentiviral vector (45). The efficiency of LANA depletion was determined by comparing the levels of LANA with those of the control shRNA transduced cells, which showed a significant reduction in shLANA transduced cells ( Fig.…”

Section: Fig 6 Legend (Continued)mentioning

confidence: 99%

Minichromosome Maintenance Proteins Cooperate with LANA during the G ₁ /S Phase of the Cell Cycle To Support Viral DNA Replication

Dabral

Uppal

Rossetto

et al. 2019

J Virol

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Latency-associated nuclear antigen (LANA) is essential for maintaining the viral genome by regulating replication and segregation of the viral episomes. The virus maintains 50 to 100 episomal copies during latency and replicates in synchrony with the cellular DNA of the infected cells. Since virus lacks its own replication machinery, it utilizes the cellular proteins for replication and maintenance, and LANA has been shown to make many of these proteins available for replication by directly recruiting them to the viral origin of replication within the terminal repeat (TR) region. Our studies identified members of the minichromosome maintenance (MCM) complex as potential LANA-interacting proteins. Here, we show that LANA specifically interacts with the components of the MCM complex, primarily during the G1/S phase of the cell cycle. MCM3 and -4 of the MCM complex specifically bound to the amino-terminal domain, while MCM6 bound to both the amino- and carboxyl-terminal domains of LANA. The MCM binding region in the N-terminal domain mapped to the chromatin binding domain (CBD). LANA with point mutations in the carboxyl-terminal domain identified an MCM6 binding domain, and overexpression of that domain (amino acids [aa] 1100 to 1150) abolished TR replication. Introduction of a peptide encompassing the LANA aa 1104 to 1123 reduced MCM6 association with LANA and TR replication. Moreover, a recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV) expressing LANA with a deletion of aa 1100 to 1150 (BAC16Δ1100–1150, where BAC is bacmid) showed reduced replication and persistence of viral genome copies compared to levels with the wild-type BAC16. Additionally, the role of MCMs in viral replication was confirmed by depleting MCMs and assaying transient and long-term maintenance of the viral episomes. The recruitment of MCMs to the replication origins through LANA was demonstrated through chromatin immunoprecipitation and isolation of proteins on nascent replicated DNA (iPOND). These data clearly show the role of MCMs in latent DNA replication and the potential for targeting the C-terminal domain of LANA to block viral persistence. IMPORTANCE LANA-mediated latent DNA replication is essential for efficient maintenance of KSHV episomes in the host. During latency, virus relies on the host cellular machinery for replication, which occurs in synchrony with the cellular DNA. LANA interacts with the components of multiple cellular pathways, including cellular replication machinery, and recruits them to the viral origin for DNA replication. In this study, we characterize the interactions between LANA and minichromosome maintenance (MCM) proteins, members of the cellular replication complex. We demonstrated a cell cycle-dependent interaction between LANA and MCMs and determined their importance for viral genome replication and maintenance through biochemical assays. In addition, we mapped a 50-amino acid region in LANA which was capable of abrogating the association of MCM6 with LANA and blocking DNA replication. We also detected LANA along with MCMs at the replication forks using a novel approach, isolation of proteins on nascent DNA (iPOND).

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KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

Cited by 15 publications

References 70 publications

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Epigenetic control in Kaposi sarcoma-associated herpesvirus infection and associated disease

Minichromosome Maintenance Proteins Cooperate with LANA during the G ₁ /S Phase of the Cell Cycle To Support Viral DNA Replication

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KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

Cited by 15 publications

References 70 publications

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Epigenetic control in Kaposi sarcoma-associated herpesvirus infection and associated disease

Minichromosome Maintenance Proteins Cooperate with LANA during the G 1 /S Phase of the Cell Cycle To Support Viral DNA Replication

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Minichromosome Maintenance Proteins Cooperate with LANA during the G ₁ /S Phase of the Cell Cycle To Support Viral DNA Replication