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Title: Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

Abstract

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.

Authors:
;  [1];  [2];  [1]
  1. Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)
  2. Central Instrumentation Laboratory Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka 565-0871 (Japan)
Publication Date:
OSTI Identifier:
22581702
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 496; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIGENS; CENTROMERES; CHROMOSOMES; ELECTRON MICROSCOPY; FLUORESCENCE; GENES; MITOSIS; SARCOMAS; TELOMERES

Citation Formats

Rahayu, Retno, Ohsaki, Eriko, Omori, Hiroko, and Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes. United States: N. p., 2016. Web. doi:10.1016/J.VIROL.2016.05.020.
Rahayu, Retno, Ohsaki, Eriko, Omori, Hiroko, & Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes. United States. doi:10.1016/J.VIROL.2016.05.020.
Rahayu, Retno, Ohsaki, Eriko, Omori, Hiroko, and Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp. 2016. "Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes". United States. doi:10.1016/J.VIROL.2016.05.020.
@article{osti_22581702,
title = {Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes},
author = {Rahayu, Retno and Ohsaki, Eriko and Omori, Hiroko and Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp},
abstractNote = {In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.},
doi = {10.1016/J.VIROL.2016.05.020},
journal = {Virology},
number = ,
volume = 496,
place = {United States},
year = 2016,
month = 9
}
  • Retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), was first identified in retroperitoneal fibromatosis (RF) tumor lesions of macaques with simian AIDS. We cloned and sequenced the ORF73 latency-associated nuclear antigen (LANA) of RFHVMn from the pig-tailed macaque. RFHVMn LANA is structurally analogous to KSHV ORF73 LANA and contains an N-terminal serine-proline-rich region, a large internal glutamic acidic-rich repeat region and a conserved C-terminal domain. RFHVMn LANA reacts with monoclonal antibodies specific for a glutamic acid-proline dipeptide motif and a glutamic acid-glutamine-rich motif in the KSHV LANA repeat region. Immunohistochemical and immunofluorescence analysismore » revealed that RFHVMn LANA is a nuclear antigen which is highly expressed in RF spindloid tumor cells. These data suggest that RFHV LANA is an ortholog of KSHV LANA and will function similarly to maintain viral latency and play a role in tumorigenicity in macaques.« less
  • KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHC I) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER.more » By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance.« less
  • During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.
  • The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence inmore » chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes.« less
  • Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANAmore » in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes.« less