KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA

Ponnusamy, Rajesh; Petoukhov, Maxim V.; Correia, Bruno E.; Custódio, Tânia F.; Juillard, Franceline; Tan, Min; Miranda, Marta Pires de; Carrondo, Maria Arménia; Simas, J. Pedro; Kaye, Kenneth M.; Svergun, Dmitri I.; McVey, Colin E.

doi:10.1093/nar/gkv987

Cited by 14 publications

(35 citation statements)

References 73 publications

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“…LANA-Imposed DNA Bending and Nucleosome Translational Positioning Are Predicted to Direct TR Chromatin Folding. Although the DNA of each TR is known to be bent by LANA dimer binding(15,43,44), and to be occupied by four nucleosomes(31), A B p2TR tethers show two distinct clusters of LANA Ab that form a 46°angle between their long axes. (A) Compilation of emission data from 28 p2TR tethers; emissions are rendered as in Fig.…”

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confidence: 99%

Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

Grant

Loftus

Stoja

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the 3D structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C termini to the tandem array of episomal terminal repeats (TRs) and with their N termini to host chromatin. Superresolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and extends previously established findings from epifluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

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confidence: 99%

Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

Grant

Loftus

Stoja

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Given the spatial proximity of these two functional regions, it is possible that the introduction of certain mutations in the SOCS box impact DNA recognition helix ␣2, disrupting the DNA binding interface and, consequently, mLANA-mediated episome persistence. Of note, the C-terminal kLANA structure corresponds very closely to that of mLANA, and C-terminal kLANA residues 1085 to 1100 also contain a SOCS box component (5,25,52,53). The mLANA residues mutated in this work correspond to identical residues in kLANA within this motif and are similarly located near the DNA binding surface and dimerization interface.…”

Section: Discussionmentioning

confidence: 60%

Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

Cerqueira

Tan

et al. 2016

J Virol

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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 (EC 5 S mLANA ) complex and mLANA's E3 ligase activity on host NF-B 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. A s obligatory intracellular parasites, viruses have evolved mechanisms to modulate ubiquitination, which is an essential regulatory mechanism in eukaryotes, controlling a wide range of cellular pathways. Ubiquitination occurs through a threeenzyme cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase enzyme (1). E3 ligases bind to the E2-ubiquitin intermediate and the substrate, catalyzing the transfer of ubiquitin to the substrate target lysine. Many E3 ligases have been described, such as Cullin 5-RING E3 ligases (CRL5), also known as Elongin BC/Cullin 5/SOCS (EC 5 S) E3 ligases. They are multisubunit complexes containing a scaffold protein (Cullin 5) attached to a RING finger protein (Rbx) (Cullin 5-Rbx module), an adaptor heterodimer (Elongin B/C), and a substrate recognition protein (suppressor of cytokine signaling [SOCS] box protein). The last component bridges the substrate of ubiquitination and the E3 ligase complex by interacting with Elongin B/C and Cullin 5 through a SOCS box motif (2-4). 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 virusCertain viruses encode proteins with SOCS box motifs to hijack the components of cellular E3 ligases, thus modulating the ubiquitination pathway. Examples are latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) (5, 6) and murid he...

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“…Each TR contains three adjacent LANA binding sites. C-terminal LANA forms multimers through dimer-dimer interactions (43)(44)(45)(46)(47), and it is possible that a higher-order LANA structure may compensate for the segregation defect induced by deletion of amino acids 33 to 194. Since it is unlikely virus could undergo such TR recombination events and remain viable, these segregation deficiencies could be highly detrimental to KSHV in vivo.…”

Section: Discussionmentioning

confidence: 99%

Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance

Juillard

Vázquez

Tan

et al. 2019

J Virol

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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.IMPORTANCEKSHV 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.

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KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA

Cited by 14 publications

References 73 publications

Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance

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