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Manuscript title: The putative U94 integrase is dispensable for human
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herpesvirus 6 (HHV-6) chromosomal integration
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Nina Wallaschek1, Annie Gravel2, Louis Flamand2,3, Benedikt B. Kaufer1,*
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1Institut
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14163 Berlin, Germany
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2 Division
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Quebec city, Quebec G1V 4G2, Canada
für Virologie, Freie Universität Berlin, Robert von Ostertag-Straße 7-13,
of Infectious Disease and Immunity, CHU de Québec Research Center,
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Medicine, Université Laval, Quebec city, Québec,G1V 0A6 Canada
Department of microbiology, infectious disease and immunology, Faculty of
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*Corresponding author:
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Benedikt B. Kaufer, Institut für Virologie, Freie Universität Berlin, Robert von
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Ostertag-Straße 7-13, 14163 Berlin, Germany.
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Phone: +49-30-838-51936, FAX: +49-30-838-51847, e-mail: b.kaufer@fu-
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berlin.de
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Running title: Role of the U94 protein in HHV-6 integration
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Key words: Human herpesvirus 6; integration; telomeres; latency; integrase; U94
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Word count text: 1144
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Abstract
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Human herpesvirus 6 (HHV-6) can integrate its genome into the telomeres of
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host chromosomes and is present in the germline of about 1 % of the human
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population. HHV-6 encodes a putative integrase U94 that possesses all
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molecular functions required for recombination like DNA-binding, ATPase,
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helicase and nuclease activity and was hypothesized by many researchers to
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facilitate integration, ever since the discovery of HHV-6 integration. However,
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analysis of U94 in the virus context was hampered by the lack of reverse-genetic
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systems and efficient integration assays. Here, we addressed the role of U94 and
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the cellular recombinase Rad51 in HHV-6 integration. Surprisingly, we could
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demonstrate that HHV-6 efficiently integrated in the absence of U94 using a new
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quantitative integration assay. Additional inhibition of the cellular recombinase
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Rad51 only had a minor impact on virus integration. Our results shed light on this
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complex integration mechanism that includes factors beyond U94 and Rad51.
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Text
Human
herpesvirus
-6A
and
-6B
(HHV-6)
are
closely
related
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betaherpesviruses that infect humans early in life (Salahuddin et al., 1986). HHV-
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6B is the causative agent of roseola infantum, a febrile illness in infants, while
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disease associations of HHV-6A are not well characterized (Yamanishi et al.,
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1988). Both viruses have previously been shown to integrate its genetic material
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into telomeres of human chromosomes (Arbuckle et al., 2010, Arbuckle et al.,
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2013). This integration mechanism also allows vertical transmission of the virus
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via the germline, resulting in individuals that harbor the integrated virus in every
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single cell of their body (Daibata et al., 1998, Daibata et al., 1999, Mori et al.,
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2009, Tanaka-Taya et al., 2004, Kuhl et al., 2014, Osterrieder et al., 2014). This
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condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and is
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present in about 1 % of the human population (Pellett et al., 2012). The biological
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consequences of this condition are not well understood yet. A recent report
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indicates that iciHHV-6+ subjects are at greater risks of developing angina
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(Gravel et al., 2015). The molecular mechanism and the factors involved in HHV-
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6 integration remain completely unknown.
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HHV-6 encodes the U94 gene that contains all conserved domains of the
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Rep68 integrase of Adeno-associated virus 2 (AAV-2) (Arbuckle and Medveczky,
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2011, Thomson et al., 1991). Expression of U94 restores replication of a Rep-
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deficient AAV-2, suggesting that both proteins have similar functions (Thomson
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et al., 1994). U94 of both HHV-6A and HHV-6B has been shown to possess
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single-strand and double-strand DNA binding activity with a preference for
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TTAGGG repeats, as present in human telomeres. In addition, U94 has ATPase,
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helicase and 3’-5’ exonuclease activity against 3’ recessive ends in vitro (Trempe
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et al., 2015). Until now, the role of U94 in virus replication and integration could
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not be determined due to the lack of reverse-genetic systems and efficient
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integration assays that would allow quantification of virus integration.
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To investigate the role of U94 in HHV-6 integration, we deleted the entire
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U94 gene (ΔU94) in pHHV-6A (wt), a recently generated infectious bacterial
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artificial chromosome (BAC) clone of HHV-6A (strain U1102), using two-step
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Red-mediated mutagenesis (Fig. 1a) as published previously (Tang et al., 2010,
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Tischer et al., 2006, Tischer and Kaufer, 2012). In addition, we generated a
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revertant virus in which we restored U94 to ensure that no secondary mutations
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occurred during the mutagenesis process. Primers used for the mutagenesis are
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listed in Table S1. Recombinant BAC clones were confirmed by RFLP, DNA
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sequencing and Southern blotting using a specific digoxigenin (DIG)-labeled
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probe for U94 (Fig. 1b) as described previously (Kaufer et al., 2011). The
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recombinant viruses were subsequently reconstituted by nucleofection of JJHan
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cells with the HHV-6A BAC DNA as described previously (Nukui et al., 2015).
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Cell-free virus stocks were generated by concentrating the supernatant of highly
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infected JJHan cells and titrated by analyzing the genome copies in newly
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infected cells by qPCR. Multi-step growth kinetics (Oyaizu et al., 2012, Nukui et
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al., 2015) revealed that the ΔU94 mutant has a significant growth defect
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compared to the wt and revertant virus (Fig. 1c), suggesting that the impaired
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HHV-6A replication is either due to the abrogation of U94 expression or effects
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on the neighboring genes such as U95 caused by the deletion of the U94
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sequences.
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To determine the integration efficiency of recombinant HHV-6A viruses,
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we used our recently established integration assay (Gravel et al, submitted).
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Briefly, U2OS cells were infected with wt, ΔU94 and ΔU94rev that express GFP
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under the control of the HCMV major immediate early promoter. Infected GFP-
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positive cells were isolated 36 h post infection using a FACS AriaIII cell sorter
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(BD, San Jose), providing a pure infected cell population for our integration
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assays. To determine the integration efficiency of respective viruses, we
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performed FISH analyses on the infected U2OS cultures at day 14 post sorting
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as described previously (Kaufer et al., 2011, Kaufer, 2013). Despite the growth
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defect of ΔU94 during lytic replication, the integration frequency of ΔU94 in
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metaphase chromosomes was comparable to wt and revertant virus (Fig. 2a-b);
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for this purpose 90 metaphases for each virus were imaged and the proportion
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(%) determined that showed a specific signal for HHV-6 at the ends of
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chromosomes in two chromatids. To confirm that this is not a metaphase specific
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effect, we analyzed interphase nuclei for the presence of the virus genome.
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ΔU94 was detected in comparable proportion of nuclei (Fig. 2c). These findings
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indicate that U94 is not essential for HHV-6A integration. Furthermore, we
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performed qPCR analyses at day 0 and 14 post sorting to determine if the
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maintenance of the HHV-6A genome was altered upon abrogation of U94. Viral
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genome copies were quantified by qPCR relative to the cellular genome, using
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primers and probes specific for HHV-6A U86 and the cellular ß2M gene,
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respectively (Table S1). The HHV-6A genome was maintained in ΔU94 infected
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cells at comparable levels as wt and revertant viruses (Fig. 2d). Furthermore,
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clonal cell lines for wt, ΔU94 and ΔU94rev were generated to confirm the
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integration into host telomeres (Figure 3).
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To exclude that the alternative lengthening of telomeres (ALT) activity
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present in U2OS cells can compensate for the absence of U94, we confirmed our
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observations in ALT negative 293T cells (Figure S1) and JJHan cells (data not
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shown). Furthermore, no integration defect was observed in a HHV-6A mutant
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virus in which U94 expression was abrogated by insertion of multiple stop codons
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within the open reading frame (Figure S2), while viruses that lacked the viral
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telomere sequences had a severe integration defect (Wallaschek et al.,
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submitted). Taken together, our data demonstrates that U94 is not essential for
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HHV-6A integration in U2OS, 293T and JJHan cells.
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Next, we investigated if the cellular recombinase Rad51 that is essential
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for homologous recombination in mitotic cells (San Filippo et al., 2008) is
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involved in the integration of HHV-6A and could facilitate integration in the
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absence of U94. Therefore, we performed in vitro integration assays with wt and
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ΔU94 mutant viruses in the presence or absence of the specific Rad51 inhibitor
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RI-1. We used 5 µM of RI-1 as this concentration was previously shown to
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efficiently inhibit Rad51 (Budke et al., 2012), while no cytotoxic effects were
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detectable (Figure S3). Both wt and the ΔU94 mutant were still able to integrate
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into U2OS cells. A slightly decreased frequency in metaphases and interphases
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was observed (Fig. 2e and f), but our data suggest that Rad51 does not play a
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major role in the integration of HHV-6A in U2OS cells. Comparable results were
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obtained with the commercial Rad51 inhibitor B02 (data not shown). Similarly,
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qPCR analyses revealed a minor reduction in HHV-6A genome copies in Rad51
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treated cells (Fig. 2g). Taken together, our data demonstrates that both U94, the
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putative viral integrase, and Rad51, the cellular recombinase, are dispensable for
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the integration of HHV-6A into host chromosomes in the two tested cell lines,
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suggesting that other viral or cellular recombinases can complement their
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functions. Most likely, several redundant pathways ensure that HHV-6 integration
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can occur. Besides homologous recombination (HR), also single strand
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annealing (SSA) is a possible mechanism that could accomplish virus integration.
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In this scenario, cellular factors such as Rad52 or viral factors including U41 and
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U70, the homologs of HSV-1 UL12 and ICP8, which were shown to mediate
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strand exchange (Reuven et al., 2003, Schumacher et al., 2012), could also
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facilitate HHV-6 integration and are targets of future studies. In addition, we will
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established a quantitative integration assay for primary cells to assess the role of
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U94 in cells ex vivo.
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Acknowledgements
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We are grateful to Yasuko Mori (Kobe University, Japan) for providing the HHV-
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6A BAC and Ann Reum for her technical assistance. This study was supported
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by the DFG grant KA 3492/1-1 and the ERC starting grant StG-677673 awarded
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to BK.
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Figure legends
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Figure 1: Generation and characterization of the ΔU94 mutant and ΔU94
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revertant. (a) Schematic representation of the HHV-6A wt, the ΔU94 mutant and
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the ΔU94rev. (b) Southern blot analysis after HindIII digestion of the indicated
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BAC clones using a DIG-labeled U94 probe. (c) Growth kinetics comparing
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replication properties of HHV-6A wt, ΔU94 mutant and ΔU94rev virus in JJHan
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cells. HHV-6A genome copy numbers were determined by qPCR. Copy numbers
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per 1x 106 cells are shown as means of three independent experiments with
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standard errors. Significant differences between wt and ΔU94 as well as ΔU94
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and ΔU94rev (Mann-Whitney U-test, p < 0.05 or p < 0.01) are indicated with
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asterisks (* or **). dpi: days post infection.
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Figure 2: Integration efficiency and genome maintenance of the ΔU94
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mutant in the U2OS integration system. (a) Representative metaphase images
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of HHV-6A wt, ΔU94 and ΔU94rev virus in U2OS cells at d14 day post sorting.
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Cells were fixed, metaphase spreads were prepared, hybridized with a HHV-6A
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specific DIG-labeled probe, washed and incubated with a secondary anti-DIG-
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FITC antibody, DAPI was used to visualize the nuclei and chromosomes. Blue:
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DNA, green: HHV-6A genome. Scale bar corresponds to 10µm. (b and e)
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Integration frequency of indicated viruses in the absence (b) and presence of the
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specific Rad51 inhibitor RI-1 (e) was quantified by analyzing the integration
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status of at least 60 metaphases. Results are shown as means of two (e) or three
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(b) independent experiments with standard errors. (c and f) >200 interphase
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nuclei of cells infected in the absence (c) and presence of the specific Rad51
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inhibitor RI-1 (f) with indicated viruses were examined for the presence of HHV-
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6A. Results are shown as means of two (f) or three (c) independent experiments
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with standard errors. (d and g) Maintenance of the HHV-6A genome in the
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absence (d) and presence of the specific Rad51 inhibitor RI-1 (g) was
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determined by qPCR analysis at d0 and d14 post sort. Copy numbers per 1x 10 6
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cells are shown as means of three independent experiments with standard
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errors. Significant differences between ΔU94 and ΔU94 + RI-1 (Mann-Whitney U-
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test, p < 0.05) are indicated with an asterisk (*).
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Figure 3: Generation of clonal U2OS cell lines containing wt, ΔU94 and
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ΔU94rev virus. The HHV-6A genome was detected using a specific DIG-labeled
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probe by FISH (green) and nuclei and chromosomes were visualized using DAPI
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(blue). Representative images of clonal U2OS cell lines for wt, ΔU94 and
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ΔU94rev are shown. Metaphases are shown on the left, interphase nuclei on the
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right. Scale bar corresponds to 10µm.
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Figure S1: Genome maintenance and integration of the ΔU94 mutant in
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293T cells. (a) Maintenance of the HHV-6A genome was determined by qPCR
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analysis at d0 and d14 post sort. Copy numbers per 1x 10 6 cells are shown as
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means of two independent experiments with standard errors. (b) Representative
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metaphase images of HHV-6A wt and ΔU94 virus in 293T cells at d14 day post
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sorting. Cells were fixed, metaphase spreads were prepared, hybridized with a
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HHV-6A specific DIG-labeled probe, washed and incubated with a secondary
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anti-DIG-FITC antibody,
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chromosomes. Blue: DNA, green: HHV-6A genome.
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Figure S2: Integration efficiency and genome maintenance of the U94stop
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mutant in the U2OS integration system. (a) Integration frequency of indicated
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viruses was quantified by analyzing the integration status of 90 metaphases.
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Results are shown as means of three independent experiments with standard
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errors. (b) 300 interphase nuclei of cells infected with indicated viruses were
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examined for the presence of HHV-6A. Results are shown as means of three
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independent experiments with standard errors. (c) Maintenance of the HHV-6A
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genome was determined by qPCR analysis at d0 and d14 post sort. Copy
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numbers per 1x 106 cells are shown as means of three independent experiments
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with standard errors.
DAPI
was used
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to
visualize
the
nuclei and
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Figure S3: Killing curve for determination of the optimal Rad51 inhibitor
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concentration. U2OS cells were treated with increasing concentrations of the
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Rad51 inhibitor RI-1 and cell viability was checked by microscopy.
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