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

v-kLANA latent infection.

<p>Viral latency in spleens of C57 BL/6 mice 14 days after i.n. infection with 10⁴ PFU of the indicated viruses. (A) Latent titers determined by co-culture reactivation assay (closed circles) and titers of pre-formed infectious virus by plaque assay (open circles). Circles are titers of individual mice. Bars indicate mean and dashed line shows the limit of assay detection. v-kLANA titers were significantly lower than v-WT (Mann-Whitney test). *p<0.05. (B) Quantification of viral DNA-positive cells in total splenocytes and in sorted GC B cells (CD19⁺CD95⁺GL7⁺). Data are from pools of five spleens per group. Bars are frequency of viral DNA-positive cells. Error bars indicate 95% confidence intervals. (C-E) Flow cytometry analyses. Representative FACS plots from individual mice are shown in left panels. Quantification graphs in which each point represents an individual mouse are shown at the right. Bars are mean values. Data were combined from 2 independent experiments with 5 mice in each group. (C) Total number of GC B cells (CD19⁺CD95⁺GL7⁺). NS, not significant; *p<0.05 using the Mann-Whitney test. (D) Percentage of GC B cells that were YFP positive. (E) Percentage of YFP positive cells that were GC B cells. ***p<0.001 in (D) and (E) using the Mann-Whitney test.</p

mLANA mediates kTR episome persistence.

<p>Gardella gel after transfection of A20 or A20/mLANA cells with pRepCK vector or k8TR DNA. Lanes contain 2-3x10⁶ cells. Gel was performed at 24 days of puromycin selection. Blot was probed with ³²P-pk8TR DNA. O, gel origin; E, S11 episomes; L, S11 linear genomes due to lytic replication; ccc plasmid DNA is indicated.</p

Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.

<p>Episome maintenance of mTR or kTR DNA in mLANA or kLANA expressing cells.</p

mLANA and kLANA expression in vivo.

<p>Spleen sections of mice infected with 10⁴ PFU of v-WT or v-kLANA for 14 days. (A) In situ hybridization (brown) with probes for viral miRNAs 1–6. Sections were counter stained with Mayer´s Haemalum. (B, C) Detection of kLANA (B) and mLANA (C) by immunohistochemistry in sections adjacent to those shown in panel A. Arrows in panel B indicate the same kLANA positive cell. Arrow in panel C indicates a mLANA positive cell. Sections were counterstained with haematoxylin. No kLANA signal was detected in sections stained only with secondary antibody. (D, E) mLANA and kLANA nuclear dots detected by indirect immunofluorescence. Images are maximum intensity projections of Z-stacks acquired over the thickness of the spleen sections. No dots were observed in unstained sections or with secondary antibody alone. Magnification 630x. (F) Quantification of mLANA (n = 69 nuclei from 3 mice) or kLANA (n = 67 nuclei from 3 mice) dots per 100 μm³ nuclear volume. Bars indicate means. The number of dots per volume was not significantly different between v-WT and v-kLANA mice (Mann-Whitney test, p>0.05). (G) Viral genomes in FACS sorted YFP⁺ and YFP^- GC B cells from spleens of v-WT.yfp (n = 7) and v-kLANA.yfp (n = 6) infected mice. Circles represent individual mice. Bars indicate means. There was no significant difference between the two infection groups (Mann-Whitney test, p>0.05).</p

kLANA mediates mTR episome persistence.

<p>(A) Schematic of kLANA and mLANA. Homologous regions are indicated in grey shading. White and black regions share no homology. Amino acid residue numbers are indicated. P, proline-rich. Gardella gels after transfection of m4TR (B) or m8TR (C) DNA. Blots in B and C were probed with ³²P-pRepCK DNA. (D) Gardella gel after 87 days of indicated cell lines from panels B and C. Blot was probed with ³²P-m8TR DNA. Days of G418 selection are below each panel. O, gel origin; ccc plasmid DNA is indicated. Lanes contain 1.5-2x10⁶ cells. Vertical lines at right (panels B, C, E) indicate positions of episomal bands. Asterisks indicate faint episomal bands. (E) Immune fluorescence for kLANA. m8TR cells are from cell line d (panels C, D). Brightness and contrast were uniformly adjusted in panels from the same field and red signal was uniformly enhanced for k8TR panels using Adobe Photoshop. Magnification, 630x.</p

Generation and lytic growth of MHV68 chimeric viruses.

<p>(A) Schematic diagram. The kLANA cassette was inserted between the M11 stop codon and the mORF72 exon in place of MHV68 103,935–104,709, which includes most of the mLANA ORF. p1, p2, p3, are mLANA promoters[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref038" target="_blank">38</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006555#ppat.1006555.ref039" target="_blank">39</a>]. The mORF72 noncoding exon (black) is located within the mLANA coding region. The E2 splice acceptor site (nt 104,871) and the mORF72 exon splice donor site (nt 104,715) were left intact to ensure expression of kLANA and mORF72. The mLANA start codon and three downstream ATGs were mutated to ATT to prevent initiation of translation (indicated by black dots). The BamHI-G fragment (genomic nt 101,653–106,902) is indicated. mLANA ORF, nt 104,868–103,927. (B) Confocal immunofluorescence detection of mLANA (top panels) or kLANA (lower panels) from yfp viruses. Magnification 630x. (C) Immunoblot of viral proteins. (D) Growth curves of virus in BHK-21 cells after infection with 0.01 PFU/cell. There was no significant difference between infection groups (p>0.05 using one-way non-parametric ANOVA Kruskal-Wallis). (E) Lung virus titers 7 days after infection with 10⁴ PFU of the indicated viruses. Circles represent titers of individual mice (n = 19). Bars indicate the mean. v-Δ1007-21.yfp had significantly lower titers than v-WT.yfp (**p<0.01, using one-way non-parametric ANOVA Kruskal-Wallis followed by Dunn´s multiple comparison test). There were no other statistically significant differences between groups.</p

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

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