Murine gammaherpesvirus 68 LANA acts on terminal repeat DNA to mediate episome persistence

Abstract

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.

Fig 1

Schematic diagrams. (A) Comparison of KSHV LANA and mLANA. Homologous regions are shown with similar shading. Unshaded regions lack homology. The C-terminal domains (darkly shaded) share the highest level of homology. C-terminal KSHV LANA mediates self-association, DNA binding, and chromosome association. N-terminal KSHV LANA (residues 1 to 32) mediates chromosome association through interaction with histones H2A and H2B and includes a nuclear localization signal. Amino acid residues are indicated. P, proline-rich region; LZ, predicted leucine zipper. (B) Schematic diagram of mLANA transcription from the MHV68 genome. MHV68 ORFs are indicated. The three potential LANA promoters, p1, p2, and p3, are indicated. Genomic nucleotide positions are indicated for the terminal repeat, the mLANA ORF, and exons 1, 2, and 3 (2, 12). When expressed from an upstream mTR element, multiple exon 1 copies are present in transcripts. A second exon (not shown) in the mTR (located at positions 119,373 to 119,195) can be driven by promoter p2 from an upstream mTR element, and a single copy of this exon is then spliced to exon 1 copies (12, 34). The line drawn above exon 3 and promoter 3 and extending into ORF75c indicates the sequence upstream of mLANA included in the native promoter constructs used in this work. (C) Schematic diagrams of mLANA and mTR constructs used in this work. Promoters are indicated. One arrow is used in the mTRs to represent the two potential promoters present in each of the mTRs (see panel A and Discussion for details). Arrows within the mTR elements indicate the direction relative to mLANA. In the native genomic MHV68 orientation, the arrow points toward mLANA. Summaries of numbers of G418-resistant cell lines positive for episomes over the total number tested, with percentages in parentheses, are shown at right for A20 and MEF cells. ND, not determined.

Fig 2

mTR DNA persists as an episome in S11 cells. m8TR or pRepCK vector was transfected into MHV68-infected S11 cells. Seventy-two hours later, cells were seeded into microtiter plates at 10,000 cells/well or 1,000 cells/well and placed under G418 selection. G418-resistant cells were expanded and assessed for episomes by use of Gardella gels. This figure is representative of two experiments. (A) Gardella gel containing S11 cells (lanes 1, 11, and 20), naked m8TR (lane 2) or pRepCK (lane 3) plasmid DNA, m8TR-transfected, G418-resistant S11 cells (lanes 4 to 10 and 12 to 19), and vector-transfected, G418-resistant S11 cells (lanes 21 and 22). A total of ∼1.5 × 10⁶ cells was loaded in each lane. G418-resistant cell lines were taken from plates seeded with 10,000 cells per well (lanes 17 to 19, 21, and 22) or 1,000 cells per well (lanes 4 to 10 and 12 to 16). The Gardella gel analysis was performed after 90 days of G418 selection. The faster-migrating signal for m8TR (lane 2) or vector (lane 3) is circular covalently closed DNA. The blot was probed with ³²P-radiolabeled pRepCK vector DNA. (B) S11 cells (lanes 1 to 3, 7 to 9, and 13 to 15) and a vector-transfected, G418-resistant S11 cell line (lanes 4 to 6, 10 to 12, and 16 to 18) that contained episomal DNA were assessed after treatment with TPA or acyclovir (ACV). The experiment was performed after 250 days of G418 selection. Probes consisted of vector (lanes 1 to 6), m8TR plasmid (which includes both mTR and vector sequences) (lanes 7 to 12), and the mLANA ORF (lanes 13 to 18). For lanes 1 to 6, 1.5 × 10⁶ cells were loaded per lane; for lanes 7 to 12, 0.15 × 10⁶ cells were loaded per lane; and for lanes 13 to 18, 1.5 × 10⁶ cells were loaded per lane. Fewer cells were used with the mTR probe due to the greater sensitivity of detection resulting from the number of repeated mTR elements in MHV68. The gel origin (O) and S11 episomal (E) and linear (L) forms (due to MHV68 lytic replication) are indicated.

Fig 3

CMV promoter-driven mLANA in cis with mTR elements persists in episomal form at low efficiency. A20 cells were transfected with plasmids containing CMV promoter-driven mLANA and mTR elements or with mTR DNA. Seventy-two hours later, cells were seeded in microtiter plates at 1,000, 100, or 10 cells/well and placed under G418 selection. A total of 2 × 10⁶ to 3 × 10⁶ A20 cells was loaded per lane in Gardella gels, and 1.5 × 10⁵ S11 cells were loaded per lane. (A) Gardella gel containing S11 cells (lane 1), naked m4TR DNA (lane 2), naked CmLANA DNA (lanes 3 and 26), naked CmLANA-m4TR plasmid DNA (lanes 4 and 25), m4TR-transfected, G418-resistant A20 cells (lanes 5 and 6), CmLANA-m4TR-transfected, G418-resistant A20 cells (lanes 7 to 20 and 27 to 32), and CmLANA-transfected, G418-resistant A20 cells (lanes 21 to 24 and 33). G418-resistant cell lines were taken from plates seeded with 1,000 cells per well (lanes 22 and 24) or 100 cells per well (lanes 5 to 21, 23, 27 to 33). (B) Gardella gel containing naked CmLANA-m4TR (lane 1), naked m2TR (lane 2), naked CmLANAF-m2TR (lane 3), naked CmLANAF (lane 4), naked CmLANAF-m8TR (lane 23), and naked m8TR (lane 24) plasmid DNA, as well as G418-resistant A20 cells transfected with CmLANAF-m4TR (lanes 5 to 8), m2TR (lanes 9 to 12), CmLANAF-m2TR (lanes 13 to 18), CmLANAF (lanes 19 to 22), CmLANAF-8TRrev (lanes 25 to 30), or m8TR (lanes 31 to 34). G418-resistant cell lines were taken from plates seeded with 100 cells per well (lanes 5 to 8, 10 to 12, 15 to 18, 20 to 22, and 30 to 34) or 10 cells per well (lanes 9, 13, 14, 19, and 25 to 29). The gel origin (O) is indicated. The number of days of G418 selection is shown below each panel. For naked plasmid DNA, the fastest-migrating signal is circular covalently closed DNA. Blots were probed with ³²P-radiolabeled m8TR DNA. Vertical lines indicate migration locations of episomal DNA.

Fig 4

mLANA levels after transfection of different expression vectors. A20 cells were transfected with the indicated plasmids and harvested for immunoblotting 72 h later, except for the sample in lane 12. The gel shows A20 cells (lanes 1 and 13) and A20 cells transfected with m4TR (lane 2), CmLANAF-m4TR (lanes 3 and 14), CmLANAF-m2TR (lane 4), CmLANAF (lane 5), m8TR (lane 6), mLANAF-m8TRrev (lane 7), mLANAF-m4TR (lane 8), mLANAF-m2TR (lane 9), mLANAF (lane 10), mLANAF-m8TR (lane 11), or CmLANAF-m8TRrev (lane 15). Lane 12 contains mLANA-m4TR cl.a, which is the G418-resistant cell line containing episomes shown in Fig. 5A, lane 8. The bottom panel shows a tubulin immunoblot for the same cells. A total of 0.25 × 10⁶ cells was loaded in each lane. The middle panel shows a shorter exposure (10 s) of lanes 8 to 12 than the 10-min exposure for these lanes in the top panel. The top right panel shows a 20-min exposure. This figure is representative of at least two experiments.

Fig 5

Native promoter-driven mLANA in cis with mTR elements persists in episomal form with increased efficiency. A20 cells were transfected with plasmids containing native promoter-driven mLANA with mTR elements or with mTR DNA. Seventy-two hours later, cells were seeded into microtiter plates at 1,000, 100, or 10 cells/well and placed under G418 selection. A total of 2 × 10⁶ to 3 × 10⁶ cells was loaded per lane for Gardella gels. (A) Gardella gel containing S11 cells (lane 1), naked m4TR plasmid DNA (lane 2), naked mLANAF-m4TR plasmid DNA (lane 3), naked mLANAF plasmid DNA (lane 4), G418-resistant, m4TR-transfected A20 cells (lanes 5 to 7), G418-resistant, mLANAF-m4TR-transfected A20 cells (lanes 8 to 21), and G418-resistant, mLANAF-transfected A20 cells (lanes 22 to 24). G418-resistant cell lines were taken from plates seeded with 1,000 cells per well (lanes 22 to 24), 100 cells per well (lanes 5 to 11 and 17 to 20), or 10 cells per well (lanes 12 to 16 and 21). (B) Gardella gel containing S11 cells (lane 1), A20 cells (lane 2), naked m4TR DNA (lane 3), naked mLANAF-m4TR DNA (lane 4), naked mLANAF DNA (lane 5), and A20 cells after 173 days of G418 selection (lanes 6 to 16). Letters above the lanes correspond to the same letters in panel A. (C) Immunoblot of mLANA G418-resistant cell lines from panel A. Lane letters correspond to the same letters as in panel A. Lanes 11 and 20 contain cells from the same G418-resistant “b” cell line. A total of 0.25 × 10⁶ cells was loaded per lane. The middle panel shows a longer exposure for lanes 18 to 22. The bottom panel shows a tubulin immunoblot. (D) Gardella gel containing naked mLANA-m8TRrev DNA (lane 1), naked m8TR DNA (lane 2), G418-resistant, mLANA-m8TRrev-transfected A20 cells (lanes 3 to 14), A20 cells (lane 15), and G418-resistant, m8TR-transfected A20 cells (lanes 16 to 23). G418-resistant cell lines were taken from plates seeded with 1,000 cells per well (lanes 3 to 21) or 100 cells per well (lanes 22 and 23). The figure is representative of at least two experiments. Numbers of days of G418 selection are indicated below the panels. The faster-migrating bands in S11 lanes are linear MHV68 genomic DNA from cells undergoing lytic infection, and the slower-migrating band is episomal MHV68. The fastest-migrating bands in naked DNA lanes are circular covalently closed DNA. Blots in panels A, B, and D were probed with ³²P-radiolabeled m8TR DNA. Vertical lines in panel D indicate episomal DNA.

Fig 6

mLANA in cis with mTR elements persists in episomal form in MEF cells. MEF cells were transfected with plasmids containing native promoter-driven mLANA in cis with mTR elements or mTR elements alone. Forty-eight hours later, cells were trypsinized, reseeded into 15-cm dishes, and placed under G418 selection. (A) Gardella gel containing naked mLANA-m4TR DNA (lane 1), naked m8TR DNA (lane 2), naked mLANA-m8TRrev DNA (lane 3), G418-resistant, mLANA-m8TRrev-transfected MEF cells (lanes 4 to 10), G418-resistant, mLANA-m4TR-transfected MEF cells (lanes 11 to 16), and G418-resistant, m8TR-transfected MEF cells (lanes 19 to 24). (B) Gardella gel containing naked mLANA DNA (lane 1), naked mLANA-m8TRrev DNA (lane 2), naked mLANA-m2TR DNA (lane 3), MEF cells (lanes 4 and 19), G418-resistant, mLANA-transfected MEF cells (lanes 5 to 8), G418-resistant, mLANA-m8TRrev-transfected MEF cells (lanes 9 to 12), G418-resistant, m8TR-transfected MEF cells (lane 13), and mLANA-m2TR-transfected MEF cells (lanes 14 to 18). (C) Gardella gel containing S11 cells (lane 1), MEF cells (lane 2), naked m4TR DNA (lane 3), naked mLANAF-m4TR DNA (lane 6), naked mLANAF DNA (lane 5), G418-resistant, m4TR-transfected MEF cells (lanes 6 and 7), G418-resistant, mLANAF-m4TR-transfected MEF cells (lanes 8 to 11), and G418-resistant, mLANAF-transfected MEF cells (lanes 12 and 13). A total of ∼1 × 10⁶ MEF cells was loaded per lane in Gardella gels. Numbers of days of G418 selection are indicated at the bottom of panels. Blots were probed with ³²P-radiolabeled m8TR DNA.

Fig 7

mLANA in cis with mTR elements in native orientation enhances episome persistence. (A) Gardella gel containing S11 cells (lane 1), A20 cells (lane 2), naked m8TR DNA (lane 3), naked mLANA F-m8TR DNA (lane 4), mLANAF (lane 5), G418-resistant, m8TR-transfected A20 cells (lanes 6 and 7), G418-resistant, mLANAF-m8TR-transfected A20 cells (lanes 8 to 21), and G418-resistant, mLANAF-transfected A20 cells (lanes 22 and 23). G418-resistant cell lines were taken from plates seeded with 1,000 cells per well (lanes 22 and 23), 100 cells per well (lanes 6 to 18), or 10 cells per well (lanes 19 to 21). A total of ∼1.5 × 10⁶ cells was loaded per lane. The number of days of G418 selection is shown at the bottom. The blot was probed with ³²P-radiolabeled m8TR DNA. (B) Immunoblot of mLANAF and tubulin for cells in panel A. Letters above lanes correspond to the same letters in panel A.

Fig 8

mLANA concentrates to dots along mitotic chromosomes in the presence of episomes but is broadly distributed along chromosomes in episome-deficient cells. mLANAF was detected in MEF cells deficient for episomes (A to D; cells from Fig. 6C, lane 10) or in MEF cells containing mLANAF-m4TR episomes (E to H; cells from Fig. 6C, lane 11). Cells were in interphase (A, B, E, and F) or metaphase (C, D, G, and H). Overlay of green (mLANAF) with red (DNA) generates yellow. Panels C and D contain two mitotic cells. The two paired dots in panels C and D are likely due to the presence of rare episomes that were not detected by the Gardella gel. Brightness and contrast were uniformly adjusted for some panels from the same field by use of Adobe Photoshop. Magnification, ×630.