The MHV68 TR elements act in cis to mediate episome persistence.
Since the TR elements are the cis-acting sequence for KSHV episome maintenance, we assessed whether the mTR elements are the cis-acting element for MHV68. KSHV LANA acts on the KSHV TR elements to mediate episome persistence. mLANA is homologous to LANA () and is critical for establishment of viral latency in mice. We hypothesized that the mTR elements might have a role in episome maintenance similar to that of the KSHV TRs.
We assayed whether MHV68 TR DNA can mediate extrachromosomal persistence of DNA in murine B lymphoma S11 cells. Most S11 cells are latently infected with MHV68, although some S11 cells contain virus undergoing lytic replication. In latently infected cells, MHV68 persists as a multicopy episome (61
). Therefore, the necessary factors responsible for MHV68 episome persistence are expressed in these cells. If the mTRs are the cis
-acting sequence for episome persistence, then DNA containing the mTR sequence is expected to be capable of persisting as an episome in S11 cells. m8TR (contains eight copies of the MHV68 TR element), m4TR (contains four copies of mTR), or vector alone was transfected into S11 cells. Cells were then seeded into microtiter plates at 10,000 or 1,000 cells per well and placed under G418 selection, for which resistance is encoded on the plasmid. In at least three experiments, outgrowth of G418-resistant cells transfected with m8TR was robust and occurred in all 96 wells in the plates seeded with 10,000 cells per well and in an average of 20 wells in the plates seeded with 1,000 cells per well. Outgrowth was also robust in cells transfected with m4TR, and an average of 86 wells were positive for outgrowth at 10,000 cells per well and 7 wells were positive at 1,000 cells per well. In contrast, after transfection with the vector control, which lacks mTR elements, outgrowth occurred in only an average of 9 wells and 1 well after seeding at 10,000 and 1,000 cells per well, respectively. These outgrowth data indicate that mTR DNA persists more efficiently than the vector in S11 cells. The observed enhanced persistence could be due to maintenance of mTR DNA as episomes or to increased efficiency of integration of mTR DNA.
To assess the presence of m8TR episomal DNA, Gardella gel analyses were performed on G418-resistant cell lines. In Gardella gels (29
), live cells are loaded into gel wells and lysed immediately at the start of the gel run. Episomes as large as several hundred kilobases can migrate into the gel, while chromosomal DNA remains at the gel origin. Episomes are then detected by Southern blotting. S11 cells (, lanes 1, 11, and 20) contain episomes but were not detected because the probe consisted of vector only, which does not share sequence with MHV68. After 90 days of G418 selection, m8TR episomes were detected in 11 (, lanes 4, 6 to 10, 14, 15, and 17 to 19) of 16 lanes. In a total of two experiments, 26 of 35 G418-resistant cell lines contained episomes. In addition, three of six m4TR-transfected, G418-resistant cell lines had episomes (data not shown). The episomes generally migrated much slower than covalently closed circular plasmid DNA (, lane 2, faster-migrating band), consistent with recombination events. Selection of enlarged KSHV episomes is frequently observed in KSHV episome maintenance assays and is due to recombination events resulting in increased numbers of TR elements in episomes. Therefore, MHV68 TR DNA can persist as episomes in cells latently infected with MHV68.
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. (more ...) Integration of transfected DNA into MHV68.
Unexpectedly, one of the two G418-resistant cell lines transfected with vector, which lacks TR sequence, had large amounts of extrachromosomal DNA (, lane 22). In fact, extrachromosomal signals in vector-transfected S11 cells were observed for 3 of 6 G418-resistant cell lines tested in a total of two experiments. Since the vector lacks mTR DNA and any MHV68 sequence, it was expected that integration into host cell chromosomes would be required to allow persistence of DNA and that transfected DNA would not persist as an episome. Notably, two of the G418-resistant cell lines containing m8TR episomes (, lanes 8 and 10) also had large amounts of extrachromosomal DNA, which ran in a similar pattern on the gel compared with that of the vector-transfected cell line (, lane 22). Of the 35 G418-resistant cell lines transfected with m8TR, this finding of large amounts of extrachromosomal DNA was observed in 5 cell lines and in 1 of 6 G418-resistant cell lines transfected with m4TR ( and data not shown).
One possible explanation for the vector being maintained as an episome was that the vector had integrated into MHV68 genomes, which are episomal. To test this possibility, the Southern blot was stripped of signal and reprobed for a sequence present in MHV68 but not the transfected mTR DNA. Strikingly, overlay of the two films demonstrated complete signal overlap for the cell line containing extrachromosomal vector (data not shown). Notably, the two cell lines containing m8TR DNA in a pattern similar to that of the extrachromosomal vector noted above also had a confluence of signals when the films were overlaid (data not shown). These results are consistent with integration of vector and m8TR DNAs into MHV68 genomes in these G418-resistant cell lines.
Since the integration of vector lacking any MHV68 DNA into MHV68 episomal genomes was an unexpected event, we further investigated this finding. S11 cells or a G418-resistant S11 cell line transfected with vector (lacking mTR DNA) but containing extrachromosomal vector DNA was assessed after 250 days of G418 selection. Cells were treated with 20 ng/ml tetradecanoyl phorbol acetate (TPA) or 100 μM acyclovir for 48 h. TPA induces lytic infection, while acyclovir inhibits lytic infection. As expected, probing with vector did not detect a signal in S11 cells (, lanes 1 to 3), since the probe lacks MHV68 sequence. However, the vector probe detected episomal (E) (, lane 4) and linear (L) (, lane 4) DNA (from lytic infection) in the G418-resistant cells containing vector episomal DNA. Incubation with TPA increased the vector-containing linear, replicating DNA signal (, lane 5) but not the episomal signal, which is the expected effect on S11 virus, since TPA induces lytic replication. In contrast, treatment with acyclovir decreased the amount of linear, replicating DNA but not the episomal signal (, lane 6), which is the expected effect on MHV68 infection. A Gardella gel with the same cell lines was also probed with mLANA, which is present in MHV68 but not in the vector, to detect MHV68 DNA. In both S11 cells and G418-resistant, vector-transfected S11 cells, similar patterns of increasing linear DNA after TPA treatment and decreased linear DNA after acyclovir treatment were observed (, lanes 13 to 18).
Simultaneous probing of a Gardella gel with sequences specific for both vector and mTRs detected the same episomal and linear replicating DNA patterns with TPA and acyclovir. No additional bands or doublets were detected in the cells containing episomal vector DNA compared with the S11 cells (, lanes 7 to 12). This finding strongly indicates that both probes detected the same bands. Overall, these findings are consistent with vector integration into MHV68 episomal genomes in some G418-resistant S11 cell lines. Furthermore, these results suggest that some G418-resistant m8TR cell lines (such as in , lanes 8 and 10) also have integration of transfected vector into episomal MHV68 genomes. Importantly, however, most G418-resistant cell lines transfected with m8TR did not have an overlap of signal when probed for mLANA compared with the vector probe. This result is therefore consistent with independent episome persistence of the transfected m8TR DNA in these lines.
CMV promoter-driven mLANA acts on mTR DNA in cis to mediate low levels of episome persistence.
Since m8TR and m4TR persisted as episomes in S11 cells, we wished to investigate whether mLANA acts on mTR DNA to mediate episome maintenance analogous to the KSHV LANA function. To test this possibility, we constructed plasmids containing CMV promoter-driven mLANA or mLANA with a C-terminal FLAG tag (mLANAF), and also plasmids containing two, four, or eight mTR elements (termed CmLANA-m2TR, CmLANAF-m2TR, CmLANA-m4TR, CmLANAF-m4TR, or CmLANAF-m8TRrev). As controls, we generated plasmids containing only mLANA (termed CmLANA or CmLANAF) or used m2TR, m4TR, and m8TR, which contain only TR elements. Uninfected mouse A20 B lymphoma cells were transfected with m2TR, m4TR, m8TR, CmLANA, CmLANAF, CmLANA-m2TR, CmLANAF-m2TR, CmLANA-4TR, CmLANAF-4TR, or CmLANAF-m8TRrev. Cells were seeded into microtiter plates at 3 days posttransfection and placed under G418 selection, against which resistance is encoded by the plasmid vector. G418-resistant cell outgrowth was similar for each of the transfections containing mTR DNA, whether or not mLANA was present in cis. After transfection with mTR-containing DNA, nearly all 96 wells were positive for outgrowth in plates seeded at 1,000 cells/well, ~10 to 30 wells were positive after seeding at 100 cells per well, and ~0 to 5 wells were positive after seeding at 10 cells per well. G418-resistant cell outgrowth for the CmLANA or CmLANAF transfections was lower, at ~50 positive wells for plates seeded at 1,000 cells/well, ~10 positive wells after seeding at 100 cells per well, and ~0 to 2 wells after seeding at 10 cells per well. The comparable rates of G418-resistant cell outgrowth with mTR transfections, regardless of the presence of mLANA, were consistent with either an absence of mLANA episome maintenance or episome maintenance occurring at a rate similar to that of integration.
G418-resistant cells were expanded and assessed by Gardella gel analysis for the presence of episomes. m2TR-transfected cells (, lanes 10 to 13), m4TR-transfected cells (, lanes 5 and 6), and m8TR-transfected cells (, lanes 31 to 34), which contain mTR elements but not the mLANA sequence, did not contain episomes. Similarly, cells transfected with CmLANA (, lanes 21 to 24 and 33) or CmLANAF (, lanes 19 to 22), which contain the mLANA sequence but do not have mTR elements, also did not contain any episomes. Therefore, no episomes were present when either mLANA was present without mTR elements or mTR elements were present without mLANA.
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 (more ...)
G418-resistant cells transfected with DNA containing both mLANA and mTRs were also assessed for the presence of episomes. CmLANAF-m2TR, which contains FLAG-tagged mLANA and two mTRs, had no episomes in six G418-resistant cell lines (, lanes 13 to 18). Similarly, cells transfected with CmLANA-m2TR, which contains mLANA without an epitope tag and two mTRs, had no episomes in four G418-resistant cell lines (data not shown). However, CmLANA-m4TR, which contains mLANA and four mTR elements (, lanes 7 to 20 and 27 to 32), had episomal DNA in four lanes (, lanes 9, 27, 28, and 30), and in a total of two experiments, CmLANA-m4TR-transfected cells had episomes in 9 of 33 (27%) G418-resistant cell lines. In addition, CmLANAF-m4TR, which contains FLAG-tagged mLANA and four mTR elements, had episomal DNA in one lane (, lane 5), and in a total of two experiments, CmLANAF-m4TR had episomes in 2 of 24 (8%) G418-resistant cell lines. CmLANAF-m8TRrev had episomes in 1 (, lane 29) of 18 (6%) G418-resistant cell lines (, lanes 25 to 30, and data not shown). Therefore, when mLANA expressed from a CMV promoter was present in cis with 4 or 8 mTR elements, episomes were present in a small percentage of G418-resistant cell lines. This finding is consistent with mLANA acting on mTR elements to mediate low-efficiency episome persistence.
The native mLANA promoter drives higher-level mLANA expression than the CMV promoter.
We reasoned that a low expression level of mLANA might have been responsible for the low efficiency of episome persistence and therefore assessed mLANA expression levels driven by either the CMV promoter or the native mLANA promoter. CmLANAF, CmLANAF-m2TR, CmLANAF-m4TR, and CmLANAF-m8TRrev, which have mLANA driven by the CMV promoter, were transfected into A20 cells and analyzed for mLANA expression by immunoblotting with anti-FLAG antibody. CmLANAF-m4TR (, lanes 3 and 14) expressed mLANA at a higher level than did CmLANAF (, lane 5) or CmLANAF-m2TR (, lane 4). CmLANAF-m4TR mLANA expression (, lanes 3 and 14) was also higher than that of CmLANAF-m8TRrev (, lane 15). It is possible that the higher expression level from CmLANAF-m4TR accounted for its higher efficiency of episome persistence than that of CmLANAF-m2TR or CmLANAF-m8TRrev ().
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 (more ...)
mLANAF, mLANAF-m2TR, mLANAF-m4TR, and mLANAF-m8TRrev each have mLANA driven by its native promoter sequence and were also tested for mLANA expression levels. Three native promoters have been described for mLANA, including one immediately upstream of mLANA and two within the mTR elements (2
). The orientation of the mTR elements in relation to mLANA in mLANAF-m2TR and mLANAF-m4TR is the same as in genomic MHV68, but it is reversed in mLANAF-m8TRrev. mLANAF (, lane 10) expressed mLANA at a similar level to those of mLANAF-m8TRrev (, lane 7) and CmLANAF (, lane 5). mLANAF-m2TR (, lane 9) and mLANAF-m4TR (, lane 8) expressed mLANA at substantially higher levels than those of mLANAF and mLANAF-m8TRrev. The expression level for mLANAF-m4TR was higher than that for mLANAF-m2TR, as evident in the 10-s exposure (, middle panel, lanes 8 and 9, respectively). Therefore, the presence of upstream mTR elements in the native genomic orientation relative to mLANA substantially enhances mLANA expression, and the level of expression is much greater than that driven by the CMV promoter. Furthermore, four upstream mTR elements were more efficient than two mTR elements in driving mLANA expression.
Native promoter-driven mLANA maintains mTR episomes with enhanced efficiency.
Since higher mLANA expression occurs from the native promoter than from the CMV promoter, we assessed whether episome maintenance is enhanced when native promoter-driven mLANA is oriented in cis with mTR elements. mLANAF, m4TR, and mLANAF-m4TR were each transfected into A20 cells, seeded into microtiter plates at a density of 1,000 cells per well, 100 cells per well, or 10 cells per well, and placed under G418 selection. For m4TR and mLANAF-4TR, G418-resistant outgrowth occurred in nearly all wells seeded at 1,000 cells per well, in ~15 wells seeded at 100 cells/well, and in ~2 wells seeded at 10 cells/well. This pattern of similar G418-resistant cell outgrowth after transfection of all mTR-containing DNA constructs in both the presence and absence of mLANA continued in subsequent experiments. In comparison, mLANAF G418-resistant cell outgrowth occurred in only ~30 wells seeded at 1,000 cells/well, ~2 wells seeded at 100 cells/well, and 0 wells seeded at 10 cells/well. The higher rate of G418-resistant cell outgrowth for cells transfected with mTR-containing DNA than that for cells in the absence of mTR DNA provided evidence for a higher rate of integration of mTR-containing plasmids into the host genome. Since there was no increase in the G418 outgrowth rate with mLANA and mTRs expressed in cis, results were consistent with either a lack of episome persistence or an efficiency of episome persistence that was no higher than that of integration.
G418-resistant cell lines were expanded and assessed for the presence of episomes by Gardella gel analysis at 47 days postselection. As expected, neither m4TR (, lanes 5 to 7), which lacks mLANA, nor mLANAF (, lanes 22 to 24), which lacks mTR elements, had episomes. In contrast, mLANAF-m4TR persisted as episomal DNA in 10 (, lanes 8 to 10, 12 to 16, 19, and 21) of 14 lanes. Notably, most of the episomal signal migrated much more slowly than the covalently closed circular mLANAF-m4TR plasmid (, lane 3, bottom band) and even slower than episomal MHV68 from infected S11 cells (, lane 1, upper band), consistent with selection for recombination into very large episomes. In two experiments, mLANAF-m4TR had episomes in 19 of 45 (42%) G418-resistant cell lines. Furthermore, in two additional experiments, mLANA-m4TR, which is similar to mLANAF-m4TR except that mLANA lacks a C-terminal epitope tag, episomes were present in 18 of 42 (43%) G418-resistant cell lines (data not shown). Since mLANAF-m4TR persisted as episomal DNA with an efficiency similar to that of mLANA-m4TR, the C-terminal FLAG tag did not adversely affect mLANA function.
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 (more ...)
To assess the long-term stability of the episomes, selected G418-resistant cell lines were again assayed by Gardella gel analysis after 173 days of selection. All five cell lines (, lanes 8 to 14) continued to stably maintain episomal mLANAF-m4TR DNA. Therefore, episomes were stable for at least ~6 months in continuous culture.
mLANA expression was assessed in these G418-resistant cell lines. As expected, no signal was detected in m4TR (, lanes 1, 13, and 14)-transfected cell lines, since this plasmid lacks mLANA. mLANAF expression was detected in only one (, lane 21) of three (, lanes 11, 12, 20, and 21) mLANAF-transfected, G418-resistant cell lines and was expressed at a relatively low level. In contrast, mLANAF expression was detected in all mLANAF-m4TR cell lines (, lanes 2 to 10 and 15 to 18) and was robust in all lines except for one that contained very low levels of episomes (, lane 21). For this cell line, mLANAF could be detected only on longer exposure (, middle panel, lane 19). It is interesting that robust mLANAF expression (, lanes 5, 15, 16, and 18) was present even in cell lines that lacked episomes (, lanes 11, 17, 18, and 20) when the plasmid was integrated.
We observed that A20 cells expressing mLANA proliferated at a lower rate than that of cells that did not express mLANA, suggesting that mLANA may exert growth-inhibitory effects. Therefore, it is possible that when cell outgrowth is robust in nearly all wells, such as after plating 1,000 cells/well after transfection of mTR-containing plasmids, cells with integrated plasmid and lacking mLANA expression may overgrow those cells which contain episomes and express mLANA. However, in these experiments, Gardella gel analyses were generally done from plates seeded with 100 cells/well that had low levels of G418-resistant outgrowth, in which outgrowth is expected to be nearly clonal.
The presence of two or eight mTR elements instead of four mTR elements in cis with mLANA was also assessed with regard to episome maintenance. In three experiments, mLANA-m2TR (without an epitope tag) had episomes in 14 of 32 (44%) G418-resistant cell lines, while in two experiments, mLANAF-2mTR (containing a FLAG tag) had episomes in 5 of 18 (28%) cell lines (data not shown). These rates of episome persistence were very similar to those with four mTRs (42% for mLANAF-m4TR and 43% for mLANA-m4TR). mLANA-m8TRrev-transfected, G418-resistant cell lines contained episomes in only 3 (, lanes 7, 8, and 14) of 12 (, lanes 3 to 14) cell lines. In two experiments, mLANA-m8TRrev had episomes in 3 of 16 (19%) G418-resistant cell lines, and the signal was very weak in two of the lanes (, lanes 7 and 14). Two additional experiments assessing mLANAF-m8TRrev (containing an mLANA FLAG epitope tag) had episomes in 2 of 21 (10%) G418-resistant cell lines. Therefore, mLANA-m8TRrev and mLANAF-m8TRrev had lower efficiencies of episome persistence than vectors containing two or four copies of mTR. This lower efficiency was likely due to lower mLANA expression from this construct, which has the mTRs reversed from their native orientation relative to mLANA (, lane 7). In the absence of mLANA (, lanes 16 to 23), no episomes were observed. Therefore, episome persistence occurred only when mLANA and mTRs were together in cis, and episome persistence was more efficient at higher mLANA expression levels.
mLANA maintains mTR episomes in MEF cells.
Since mLANA in cis with mTRs persisted as episomal DNA in A20 B lymphoma cells, we assessed if mLANA could also maintain episomes in another cell type. Therefore, MEF cells were transfected with mLANA, mLANAF, m4TR, m8TR, mLANA-m2TR, mLANA-m4TR, mLANA-8TRrev, or mLANAF-m4TR and placed under G418 selection. G418-resistant colonies were expanded independently and assessed by Gardella gel analysis for the presence of episomes. As expected, the mLANA (, lanes 5 to 8), mLANAF (, lanes 12 and 13), m4TR (, lanes 6 and 7), and m8TR (, lanes 18 to 24, and B, lane 13) cell lines did not contain episomes. mLANA-m2TR contained episomes in 4 of 5 lanes (80%) (, lanes 14, 15, 17, and 18), mLANA-m4TR had episomes in 8 of 12 lanes (67%) (, lanes 12 to 14, 16, and 17, and data not shown), mLANA-m8TRrev contained episomes in 1 (, lane 10) of 11 lanes (9%) (, lanes 4 to 10, and B, lanes 9 to 12), and mLANAF-m4TR had episomes in 3 of 4 lanes (75%) (, lanes 8, 9, and 11). Western blot analysis of mLANAF protein expression demonstrated that the mLANAF-m4TR-transfected cell line lacking episomes (, lane 10) expressed mLANAF at a robust level that was only slightly lower than that of cells containing episomes (, lanes 8, 9, and 11), while cells transfected with mLANAF without mTRs had significantly reduced or no mLANAF expression (data not shown). These expression patterns were similar to those in A20 cells (). The lower rate of episome persistence for mLANA-m8TRrev was similar to that in A20 cells and was likely due to lower mLANA protein expression. As observed in A20 cells, MEF cells expressing mLANA proliferated at a lower rate than that of MEF cells not expressing mLANA. Therefore, mLANA acts on mTR DNA to mediate episome persistence in MEF cells.
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 (more ...) Native genomic orientation of eight mTR elements in cis with mLANA increases episome persistence efficiency.
We investigated whether the native genomic orientation of eight mTRs in cis with mLANA would affect the efficiency of episome maintenance, since mLANA-m8TRrev, in which the mTR orientation is reversed, was less efficient than mLANA in cis with only two or four mTRs in both A20 and MEF cells. Therefore, we generated mLANAF-m8TR, which contains eight mTR elements in native genomic orientation upstream of mLANA (). Transfection of mLANAF-m8TR (, lane 11) into A20 cells demonstrated substantially more mLANA expression than that of mLANAF-m8TRrev (, lane 7), and the level was somewhat higher than that of mLANAF-m4TR (, middle panel, lane 8). The expression level of mLANAF-m8TR was only slightly lower than the expression level of mLANA in a G418-resistant cell line maintaining mLANAF-m4TR episomes (, middle panel, lane 12).
We assessed the ability of mLANAF-m8TR to persist as an episome. mLANAF-m8TR, m8TR, and mLANAF were each transfected into A20 cells, plated in microtiter plates, and selected for G418 resistance. As expected, m8TR (, lanes 6 and 7) and mLANAF (, lanes 22 and 23) did not have episomes. In contrast, mLANAF-m8TR had episomal DNA in 9 (, lanes 9 to 11, 14 to 16, 18 to 19, and 21) of 15 (, lanes 8 to 21) cell lines. In two experiments, mLANAF-8TR had episomes in 13 of 18 (72%) cell lines. This episome maintenance efficiency was substantially higher than that of mLANAF-m8TRrev (10%) or mLANA-m8TRrev (19%) and was even higher than that of mLANA in native orientation with two or four mTRs in A20 cells (). Therefore, native orientation of the eight mTR elements substantially increased both mLANA expression and episome persistence.
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 (more ...)
Immunoblot analysis demonstrated that mLANA was expressed at robust levels in all mLANAF-m8TR cell lines (, lanes 3 to 9 and 13 to 19). Even cell lines lacking episomes (, lanes 3, 7 to 8, 15, and 18), which therefore contained integrated plasmid, expressed mLANA, similar to the findings with integrated mLANAF-m4TR (). Notably, cell lines transfected with mLANAF, which contains the native mLANA promoter immediately upstream of mLANA but no mTR elements, did not express mLANA (, lanes 10 and 20).
mLANA redistributes and concentrates to dots along mitotic chromosomes in the presence of episomes.
mLANA was detected in MEF cells () containing mLANAF-m4TR episomes (cell line from , lane 11) and in MEF cells deficient in episomes, for which no episomes were detected on Gardella gel analysis (cell line from , lane 10). In episome-deficient cells ( to ), mLANAF (green) was distributed broadly throughout the nucleus (red) in interphase and over mitotic chromosomes (red) (overlay of green and red generates yellow). In contrast, in the presence of episomes ( to ), mLANAF (green) was concentrated to dots both in interphase and along mitotic chromosomes (red) (overlay of green and red generates yellow). Therefore, in the presence of episomes, mLANA relocalized to dots along mitotic chromosomes.
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 , lane 10) or (more ...)