Murine Gammaherpesvirus 68 LANA Acts on Terminal Repeat DNA To Mediate Episome Persistence

Murine gammaherpesvirus 68 (MHV68) ORF73 (mLANA) has sequence homology to Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). LANA acts on the KSHV terminal repeat (TR) elements to mediate KSHV episome maintenance. Disruption of mLANA expression severely reduces the ability of MHV68 to establish latent infection in mice, consistent with the possibility that mLANA mediates episome persistence. Here we assess the roles of mLANA and MHV68 TR (mTR) elements in episome persistence. mTR-associated DNA persisted as an episome in latently MHV68-infected tumor cells, demonstrating that the mTR elements can serve as a cis-acting element for MHV68 episome maintenance. In some cases, both control vector and mTR-associated DNAs integrated into MHV68 episomal genomes. Therefore, we also assessed the roles of mTRs as well as mLANA in the absence of infection. DNA containing both mLANA and mTRs in cis persisted as an episome in murine A20 or MEF cells. In contrast, mTR DNA never persisted as an episome in the absence of mLANA. mLANA levels were increased when mLANA was expressed from its native promoters, and episome maintenance was more efficient with higher mLANA levels. Increased numbers of mTRs conferred more efficient episome maintenance, since DNA containing mLANA and eight mTR elements persisted more efficiently in A20 cells than did DNA with mLANA and two or four mTRs. Similar to KSHV LANA, mLANA broadly associated with mitotic chromosomes but relocalized to concentrated dots in the presence of episomes. Therefore, mLANA acts on mTR elements to mediate MHV68 episome persistence

Cross-species conservation of episome maintenance provides a basis for in vivo investigation of Kaposi's sarcoma herpesvirus LANA

Copyright: © 2017 Habison et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.This work was supported in part by National Institutes of Health grants CA082036 (NCI), DE025208, and DE024971 (both NIDCR), to KMK, FCT PTDC/IMI-MIC/0980/2014 to JPS, FCT Harvard Medical School Portugal Program in Translational Research (HMSP-ICT/0021/2010) to JPS, KMK, CEM, Instituto de Medicina Molecular Directors Fund to JPS, and iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) FCT/FEDER (PT2020 Partnership Agreement) to CEM. M.P.M is supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT), Portugal.info:eu-repo/semantics/publishedVersio

The Poly(A) Binding Protein Is Internalized in Virus-Induced Vesicles or Redistributed to the Nucleolus during Turnip Mosaic Virus Infection▿

Poly(A) binding protein 2 (PABP2) of Arabidopsis thaliana was previously shown to interact with VPg-Pro of turnip mosaic virus (TuMV) and may consequently play an important role during infection. Subcellular fractionation experiments revealed that PABP2 was predominantly a cytoplasmic soluble protein in healthy plants. However, in TuMV-infected plants, a subpopulation of PABP2 was membrane associated or was localized in the nucleus. Confocal microscopy experiments indicated that PABP2 was partially retargeted to the nucleolus in the presence of TuMV VPg-Pro. In addition, the membrane association of PABP2 during TuMV infection resulted from the internalization of the host protein in 6K-VPg-Pro-induced vesicles, as shown by a combination of confocal microscopy and sucrose gradient fractionation experiments. This redistribution of an important translation initiation factor to the nucleolus and to membrane structure likely underlies two important processes of the TuMV replication cycle

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome of Turnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

The RNA genome of Turnip mosaic virus is covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication

Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

AbstractTandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

3-D Microwell Array System for Culturing Virus Infected Tumor Cells

Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery

Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency

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 Å resolution. 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)