(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

['Salla Mattola', 'Department Of Biological', 'Environmental Science', 'Nanoscience Center', 'University Of Jyvaskyla', 'Jyvaskyla', 'Kari Salokas', 'Institute Of Biotechnology', 'Helsinki Institute Of Life Science', 'Hilife']

Date: 2022-06

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.

Parvoviruses are small, nonenveloped DNA viruses, that besides being noteworthy pathogens in many animal species, including humans, are also being developed as vectors for gene and cancer therapy. Canine parvovirus is an autonomously replicating parvovirus that encodes two nonstructural proteins, NS1 and NS2. NS1 is required for viral DNA replication and packaging, as well as gene expression. However, very little is known about the function of NS2. Our studies indicate that NS2 serves a previously undefined important function in chromatin modification and DNA damage responses. Therefore, it appears that although both NS1 and NS2 are needed for a productive infection they play very different roles in the process.

Funding: This work was financed by the Jane and Aatos Erkko Foundation (MVR); Academy of Finland under the award number 330896 (MVR); Biocenter Finland, viral gene transfer (MVR), and the Graduate School of the University of Jyvaskyla (SM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we used BioID approaches [ 50 – 53 , 55 ] combined with interactome-based mass spectrometry (MS) microscopy analysis [ 68 ] to investigate the nuclear interactions and nuclear localization of CPV NS2 both in noninfected and infected cells. The nuclear NS2 interactome identified by BioID included several components of different chromatin remodeling and DDR complexes. Furthermore, observations from assays with NS2 mutants suggested that N-terminal NS1/2-common splice donor mutant did not produce functional viral NS2 protein, resulting in inefficient chromatin remodeling regulation and DDR response. Altogether, our studies link CPV NS2 to novel functions in the nucleus and provide a platform for further functional analyses of NS2.

In recent years proximity-dependent biotin identification (BioID) method has been increasingly used to provide fundamental insight into the protein-protein interactions of mammalian cells. These approaches have revealed valuable details about the interactions of nuclear structures such as the nuclear pore complex (NPC) [ 50 , 51 ] and nuclear lamina [ 52 – 56 ]. Moreover, BioID also has been used to screen for proteins in cell signaling pathways [ 57 – 59 ], tight junctions [ 60 ] and on chromatin [ 61 ]. Finally, protein interactions between viruses and their hosts contributing to the outcomes of viral infections have been studied by BioID. For example, interacting partners have been identified for the Gag protein of human immunodeficiency virus type 1 (HIV-1) [ 62 – 64 ], tegument protein UL103 of cytomegalovirus [ 65 ], latent membrane protein 1 of Epstein-Barr virus [ 66 ], and Zika virus-encoded proteins [ 67 ].

The DNA damage response (DDR) machinery plays a significant role in cells by maintaining normal chromatin functions within regions of damage [ 35 ]. DDR is initiated by sensor protein-mediated detection of DNA lesions, which is followed by the activation of major signaling kinases, ataxia telangiectasia mutated (ATM), and ATM Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). This promotes signal-transduction through a series of downstream effector molecules from phosphoinositide 3 kinase-like kinases and lead to the phosphorylation of the histone H2A variant, H2AX. DDR plays a dual role in the regulation of viral replication. DDR is also involved in the intrinsic antiviral mechanisms that counter the nuclear replication of DNA viruses [ 36 – 39 ]. Conversely, DDR is also activated by many DNA viruses, and DDR factors are recruited by viruses to promote viral replication [ 39 – 42 ]. Autonomous parvovirus infection results in the induction of cellular DNA breaks and DDR activation by ATR and ATM signaling pathways [ 43 – 46 ], and it is accompanied by pre-mitotic cell cycle arrest [ 14 , 43 , 47 , 48 ]. In MVM infection viral replication is located at cellular DNA damage sites [ 34 , 49 ].

During the nuclear replication parvoviruses must either confront or embrace the chromatin remodeling machinery of the host cell. To ensure a productive infection, viruses have to recruit the cellular histone modifying and nucleosome remodeling machinery for the activation of the viral genome. Many viruses are able to counteract host-mediated silencing by recruiting and redirecting cellular histone remodeling proteins to enhance viral gene expression and replication. In herpes simplex virus 1 (HSV-1) infection the viral genome is chromatinized after its nuclear entry [ 25 ]. In a productive HSV-1 infection the modification of bound histones to an active euchromatic state is promoted by viral proteins such as ICP0 and by recruiting cellular proteins [ 26 , 27 ]. For example, the chromatin remodeling factor SNF2H (SMARCA5) protein from ISWI family complexes facilitates the transcription of viral immediately-early genes from the HSV-1 genome by removing or remodeling histones associated with viral promoters [ 28 ]. Viruses have also developed strategies for regulating host transcription by inactivating certain aspects of chromatin modeling while exploiting others to advance the viral life cycle. Most likely, manipulation of host cellular functions can be orchestrated and tuned by viral proteins. Consistent with this model, the HSV-1 single-stranded DNA-binding protein ICP8 has been found associated with cellular proteins involved in DNA replication, DNA repair, chromatin remodeling and RNA processing [ 29 , 30 ]. Similar to other DNA viruses, parvovirus replication is potentially dependent on the activation state of nucleosomes present on the nuclear viral genome [ 31 – 34 ]. The molecular mechanisms by which parvoviral proteins are involved in the interaction and epigenetic modification of nucleosomes associated with viral genomes remain poorly understood.

Autonomous parvoviruses have an ~5-kb single-stranded DNA genome encoding two viral structural proteins, VP1 and VP2, as well as two nonstructural proteins NS1 and NS2 [ 5 ]. The CPV NS2 protein is produced from the left-hand open reading frame of the viral genome and contains 87 amino-terminal amino acids that are in common with NS1 joined by mRNA splicing to 78 amino acids from an alternative open reading frame [ 6 , 7 ]. Previous knowledge on NS2 protein function in parvovirus replication is mainly derived from studies of minute virus of mice (MVM). MVM NS2 is required for efficient viral replication and capsid assembly [ 8 , 9 ]. In infected cells MVM NS2 is known to interact with two members of the 14-3-3 family of signaling proteins [ 10 ] and the survival motor neuron protein (Smn) [ 11 ]. Mutations in MVM NS2 splice acceptor or termination sites lead to severe replication defects in murine cells, whereas in other cells lines mutant viruses replicate more efficiently [ 12 , 13 ], suggesting that the requirement for MVM NS2 is cell-type specific. Moreover, MVM NS2 is required for the growth and development of viral replication centers [ 14 ]. Notably, MVM NS2 also interacts with the nuclear export factor CRM1 (also known as exportin1) [ 15 , 16 ]. CRM1 mediates the nuclear export of nuclear export signal containing proteins [ 17 – 19 ]. Although the detailed mechanisms of MVM capsid nuclear egress are still not well understood, the interaction between NS2 and CRM1 seems to be essential for the progeny virus capsid export [ 20 – 23 ]. Studies of another parvovirus closely related to CPV, feline panleucopenia virus (FPV), have shown that FPV NS2 plays a significant role in blocking pathways that promote IFN-β production, allowing the virus to evade the host antiviral innate immune response [ 24 ]. Much less is known about the role of CPV NS2 in infection. To our knowledge, the only study assessing CPV NS2 function used various NS2 mutants containing mutations and deletions that affect NS2 mRNA splicing and protein expression, or that terminate the NS2 open reading frame without altering NS1. The impact of the mutants on viral replication depended on the site of the mutation, and infection efficiency was found to be decreased with the NS2 donor mutant [ 6 ].

Autonomous parvoviruses are small single-stranded DNA viruses that depend on host cell nuclear machinery for their replication. The nuclear events of parvovirus infection include genome replication, viral assembly, and genome packaging, which require the nuclear import of structural and nonstructural viral proteins. The nonstructural protein 1 (NS1) of canine parvovirus (CPV) is a multifunctional protein with site-specific DNA binding, ATPase, nickase, and helicase activities [ 1 , 2 ], and its expression induces apoptosis in host cells [ 3 , 4 ]. NS1 is essential for initiation and direction of viral DNA replication. However, the role of the nonstructural protein 2 (NS2) in viral replication has so far remained undefined.

Results

Production of NS2 mRNA is temporally increased in infection and NS2 is localized into nucleoli Since the expression of both CPV nonstructural proteins is controlled by the P4 promoter, we were interested in examining the expression levels of NS1 and NS2 mRNA at different times post infection. The quantitative reverse transcription PCR (RT-qPCR) using specific primers that distinguished the different transcripts showed that both NS1 and NS2 mRNAs were detectable at 4 hours post infection (hpi) in NLFK cells (Fig 1A). At 6 hpi, the levels of both mRNAs were higher compared to the control 18s rRNA levels, and both continued to increase until 24 hpi. The Student’s t-test showed no statistically significant difference between the quantities of NS1 and NS2 mRNAs (p>0.05) at any time point. This data shows that NS2 is expressed early and throughout the viral replication. The early expression of NS2 is consistent with previous MVM findings [69], however, in contrast to MVM studies the expression level of CPV NS2 mRNA continued to increase during infection. MVM NS2 has a short half-life, which may account for the relatively small amount of nuclear NS2 detected in MVM infection [70]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. CPV infection leads to simultaneous expression of NS1 and NS2 genes and nucleolar accumulation of NS2. (A) Relative expression levels of NS1 (blue) and NS2 (red) measured by RT-qPCR in infected NLFK cells between 4 and 24 hours post infection (hpi). The blue and red shadings around the lines indicate the standard error of the mean (SEM, n = 3). (B) Representative confocal images of HeLa cells transfected with NS2-EGFP (green) and (C) BirA*-tagged NS2 (green) at 24 hpt in noninfected and infected cells at 24 hpi. The localization of NS1 (red) is shown in cells with DAPI-stained nucleus (gray). (D) Representative confocal images of noninfected and infected NLFK cells at 24 hpi stained with antibodies against NS2 (green) and nucleolin (red). Gray corresponds to DAPI staining of DNA. Scale bars, 3 μm. https://doi.org/10.1371/journal.ppat.1010353.g001 To characterize potential NS2 functions during CPV replication, we next examined the association of NS2 with specific sites in the host cell nucleus. Confocal microscope images showed that NS2-EGFP expressed in noninfected HeLa cells was mostly localized near the rim of the nuclear periphery and in distinct nucleolar foci (Figs 1B and S1). A similar pattern of intranuclear localization was observed in CPV-infected cells, but the nucleolar distribution of NS2-EGFP was more diffuse than in noninfected cells. The BirA*-tagged NS2 used in BioID assays localized diffusely in noninfected cells. In infected cells, the BirA*-tagged NS2 distribution was mostly similar to noninfected cells, however, some local accumulation close to the nucleoli and viral replication center identified by NS1 was detected (Fig 1C). The nucleoli were identified by the exclusion of chromatin and NS1 labels. The homogenous nuclear distribution of the BirA*-tagged NS2 is consistent with NS2 distribution in infected NLFK cells identified by antibody against NS2 (S2 Fig). The BirA*-tagged NS2 was also localized in the cytoplasm both in noninfected and infected cells, however, the expression of the recombinant protein was decreased in infected cells. As a verification of nucleolar NS2 localization, we also found that in many wild type (wt) virus infected NLFK cells the antibody-stained NS2 colocalized with the nucleoli identified by nucleolin staining (Fig 1D). These results suggested that NS2 is often accumulated to nucleoli, where some of the essential chromatin remodeling and DDR factors reside [71–74].

NS2 is associated with proteins linked to chromatin organization The NS2 BioID interactome and GO annotation analyses suggested an association between the viral NS2 protein and cellular components involved in chromatin organization. BioID based interactome linked NS2 to four different complexes which belong to the major ATP-dependent chromatin remodeling complex family ISWI (Fig 4A and S1 and S2 Tables) [75]. The mammalian ISWI complexes identified were the nucleolar chromatin remodeling complex (NoRC), WSTF-ISWI chromatin remodeling complex (WICH), the ATP-utilizing chromatin assembly and remodeling factor complex (ACF), and the remodeling and spacing factor (RSF) complex. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. NS2-associated proteins play important roles in chromatin remodeling and DDR machineries. The CPV NS2 protein is linked with certain key factors that control chromatin remodeling and DDR pathways to optimize cellular conditions for viral replication. This schematic picture shows the major associations of NS2 (red) with cellular proteins (shades of green). Dashed arrows represent key factors of chromatin binding and DDR machineries interacting with NS2. (A) The cellular chromatin modification machinery includes chromatin remodeling complexes such as NORC, WICH, ACF and RSF. (B) DNA damage response (DDR) factors contain major upstream mediators of ATM response such as DNA damage checkpoint 1 (MDC1) protein and proteins included in chromatin and nucleosome remodeling complexes. MDC1 interacts with phosphorylated γ-H2AX and mediates the recruitment of DDR response proteins to the damage site. The DDR downstream FACT complex functions as a nucleosome remodeler facilitating transcription. During replication stress, FACT orchestrates the replacement of γ-H2AX with macro-H2A.1 to the damage site. The location of parvoviral genomes and the viral replication protein NS1 (red) adjacent to DNA damage sites of the cellular chromatin are shown. Figure was created with BioRender.com. https://doi.org/10.1371/journal.ppat.1010353.g004 One of the NS2 high-confidence (BFDR <0.01) BioID hits was nucleosome-remodeling helicase matrix-associated actin-dependent regulator of chromatin A5 (gene name SMARCA5, also known as SNF2H) [76,77] which is associated with all four ISWI complexes. NS2-associated proteins included bromodomain adjacent to zinc finger domain 2A (BAZ2A, also known as TIP5, BFDR <0.01) [71,72], a regulator of SMARCA5 in NoRC. [71,72]. Notably, the highest average spectral counts in our BioID analysis were produced by the proliferation marker protein Ki-67 (MKI67; BFDR 0.02 in infected and 0.04 in noninfected cells). Ki-67, a nuclear protein expressed in actively dividing mammalian cells, is involved in the organization of heterochromatin and it also interacts with BAZ2A [78–80]. Moreover, NS2 was associated with tyrosine-protein kinase BAZ1B (also known as the Williams syndrome transcription factor, WSTF, BFDR <0.01) [81] a component of WICH with SMARCA5. NS2 was associated with both BAZ1A (BAZ1A, also known as ATP-utilizing chromatin assembly and remodeling factor 1, ACF1; BFDR <0.05) and remodeling and spacing factor 1 (RSF1, BFDR <0.01). ACF [81] and RSF [82,83,84,85] are formed when SMARCA5 combines either with BAZ1A or with RSF1. The potential role of NS2 in the regulation of transcription was further supported by its interaction with a member of the histone H2A family, core histone macro-H2A.1 (H2AFY, BFDR <0.01), which has been shown to associate with transcription repression [86]. The association between NS2 and SMARCA5, BAZ1A, BAZ1B, BAZ2A, Ki-67, and macro-H2A.1 were detected both in noninfected and infected cells. Together, these data indicate that CPV NS2 associates with proteins of four chromatin-modifying complexes. Therefore, it is possible that NS2 is involved in the manipulation of chromatin modeling processes potentially inducing modifications of both cellular and viral DNA.

NS2 is linked to DDR-associated proteins We next sought to confirm the association of NS2 with DDR factors necessary for parvoviral replication [34,87]. The progression of cell cycle is coordinated by DNA damage checkpoints, which delay or stop the cell cycle before or during DNA replication in the presence of damaged DNA [88,89]. DDR includes complex signaling cascades that require the actions of various proteins that function as DNA damage sensors, transducers, mediators, and effectors. One of the upstream mediators in DDR is DNA damage checkpoint 1 (MDC1, BFDR <0.05) protein. The BioID analysis suggested NS2 interactions with MDC1 in both noninfected and infected cells, as well as associations with DDR downstream factors, including components of the nucleolar facilitator of chromatin transcription (FACT) complex and WSTF-including nucleosome remodeling complex (WINAC) (Fig 4B and S1 and S2 Tables). FACT acts as a critical chaperon for histones in nucleosome reorganization during replication, and in the detection and response of DNA damage [90,91], stabilizing chromatin as a whole by suppressing cryptic transcription [92,93]. FACT complex subunits, structure specific recognition proteins (SSRP1, BFDR <0.01) and SUPT16H (SPT16, BFDR <0.01), were identified as high-confidence NS2 interactors both in noninfected and infected cells. SSRP1 is a histone chaperon involved in transcriptional regulation, DNA replication and damage repair [94–97]. SUPT16H also functions independently of FACT when it forms WINAC with BAZ1B (BFDR <0.01) [98,99]. WINAC is an ATP-dependent chromatin remodeling complex, which is associated with a variety of DNA processing functions. Furthermore, FACT is linked to the activation of p53, a central tumor suppressor, the stability of which is further regulated by ubiquitin carboxyl-terminal hydrolase 10 (USP10, BFDR <0.01), also identified as an NS2 interactor in BioID of noninfected cells. USP10 relocates to the nucleus in response to DDR and promotes the deubiquitination of p53 [100]. Similar function is served by E3 ubiquitin-protein ligase (TRIP12, BFDR <0.01), an NS2 interactor in infected cells, which indirectly regulates p53 activity by affecting its ubiquitination. In addition to the previously mentioned and other ubiquitination-related enzymes in the BioID results, ubiquitin-conjugating enzyme E2 N (UBE2N, BFDR <0.05) was also identified in the absence and presence of infection. It may act in non-degradation ubiquitination targeting and DNA damage repair [101]. Taken together, these results demonstrated that NS2 is associated with the DDR signaling proteins during infection. Since DDR has a clear potential role in parvovirus replication [34,87], NS2 may recruit DDR effector proteins to regulate viral replication.

Mutation of NS2 leads to changes in chromatin remodeling Our BioID analysis showed that NS2 is associated with cellular proteins involved in chromatin organization. To further validate our findings, we compared NS2 mutant clones to the wild type (wt) infectious clone to analyze their effect on chromatin organization. In these assays, cells were transfected either with the wt infectious clone or with NS2 splice donor or splice acceptor mutants (G533A and A2003T, respectively) which were designed to disrupt the expression of NS2 but not to affect the amino acid sequence of NS1 [6]. These previously described mutants are based on the MVM mutants that have been shown to alter the expression of MVM NS2 and lead to abortive infection [12,13,104]. CPV NS2 mutants have been created by introducing termination codons into the NS2 coding sequence of the infectious clone to a site upstream of the common NS1/2 splice donor or downstream of the NS2-specific splice acceptor [6]. The original publication demonstrated that the production of viral capsid proteins (VP1/VP2) and viral DNA were all significantly decreased in cells transfected with the NS2 splice donor mutant virus, while transfection with the splice acceptor mutant was comparable with wt CPV. Characterization of mutants by immunoprecipitation analyses (antibodies against the NS2 C-terminus) showed that both mutants were unable to produce intact NS2 protein [6]. However, the interpretation of mutant-induced effect on viral life cycle was complicated by RT-PCR characterization which revealed that alternative sequences used to splice the message RNA were present in both mutants [6]. It should be also noted that, that the N-terminal end of NS1, which share a common N-terminal domain with NS2, might be produced in some circumstances, and might play a role in virus-induced chromatin remodelling, although that remains to be defined. Here, we used confocal microscopy intensity analysis to study the distribution pattern and relative amount of nuclear DNA (stained with DAPI), euchromatin (labeled for H3K27ac), and heterochromatin (labeled for H3K9me3) in HeLa cells transfected with the wt CPV clone and NS2 mutants at 24 hours post transfection (hpt). Comparison of wt and mutant viral transfection revealed that the donor NS2 mutant clone displayed a clearly higher total intensity of the euchromatin (Fig 7A and 7B). It is important to note that the increased amount of euchromatin in splice donor mutant transfected cells was accompanied by significantly lower total intensity of DAPI staining (S5A Fig). This indicates that infection affects the epigenetic regulation of host chromatin, and the splice donor mutation of NS2 results in a reversion towards the decondensation state seen in noninfected cells. Distribution analyses of both nuclear DNA (S5B Fig) and euchromatin (Fig 7C) as a function of the distance from the NE further demonstrated their localization both in the nuclear periphery and in the central region of the nucleus. Additionally, transfection by all clones resulted accumulation of heterochromatin at the perinuclear region and around the nucleoli (S6A Fig). Notably, NS2 mutations did not appear to affect the relative intensity of the heterochromatin label (S6B Fig). Distribution analyses of heterochromatin further confirmed its localization both close to the nuclear periphery and in the center of the nucleus (S6C Fig). Altogether, our findings demonstrated clear changes in the quantity of euchromatin in response to NS2 donor mutant transfection, suggesting that N-terminal sequence of NS2 could have a currently undefined role in chromatin remodeling during infection. This supports our BioID results demonstrating that NS2 is associated with proteins linked to chromatin organization. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. NS2 mutation induce changes in the amount and distribution of euchromatin. (A) Representative confocal images show the nuclear localization of euchromatin marker H3K27ac (magenta), NS1 (green), and DAPI staining (gray) in nontransfected HeLa cells, and cells transfected with wt, NS2 donor and acceptor mutants at 24 hpt. (B) Total fluorescence intensities of euchromatin. (C) Nuclear distribution of H3K27ac as a function of increasing distance from the NE in nontransfected, wt and NS2 mutants transfected cells (n = 29). The error bars show the standard error of the mean. Statistical significances were determined using Dunnett’s multiple comparison test. The significance values shown are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). Scale bars, 5 μm. https://doi.org/10.1371/journal.ppat.1010353.g007

The level and localization of DDR proteins change in the presence of NS2 mutant Cellular DNA double-strand break repair sites recruit several upstream DDR proteins such as γ-H2AX [105–107] and MDC1 [108] (Fig 4B). DNA damage proteins have also been previously observed to localize to the periphery of MVM viral replication centers [109]. As shown by our BioID data, CPV NS2 is associated with MDC1 (Fig 2 and S3 Table). To further examine the role of NS2 interactions in DNA damage, we analyzed the localization and intensity of γ-H2AX and MDC1 and their connection to replication centers in HeLa cells transfected with the wt CPV, splice donor and splice acceptor mutants at 24 hpt. Our studies demonstrated that both γ-H2AX and MDC1 localized next to the replication centers and accumulated close to the nucleoli and NE in wt or the splice acceptor mutant transfected cells, in contrast to relatively diffuse nuclear localization in nontransfected cells and in cells transfected with the splice donor mutant. NS1 accumulated in distinct nuclear foci in cells transfected with the splice donor mutant (Fig 8A and 8B). As expected, both γ-H2AX and MDC1 colocalized with DNA marker. Quantitative image analysis (Fig 8C) showed that wt and the splice acceptor mutant transfections led to relatively similar amounts of nuclear γ-H2AX, whereas it was significantly decreased in nontransfected cells and in the splice donor mutant transfected cells. The distribution analyses indicated that γ-H2AX was distributed close to the NE and in the central region of the nucleus in cells transfected with the wt and the splice acceptor mutant, whereas γ-H2AX was slightly more concentrated close to the NE in nontransfected cells and in the splice donor mutant transfected cells (Fig 8D). Intensity line profiles measured through the nucleoli verified close association between γ-H2AX and replication centers in close proximity to the nucleoli both in cells transfected with the wt and the splice acceptor mutant. The close localization between replication centers and H2AX was visible to a lesser extent in the cell transfected with the splice donor mutant (Fig 8E). Similar to γ-H2AX, the total intensity of MDC1 was clearly decreased in cells transfected with the splice donor mutant in comparison to cells transfected with wt or the splice acceptor mutant (Fig 8F), and it was localized slightly more toward the NE in the splice donor mutant transfected cells (Fig 8G). The line profiles of wt and the splice acceptor mutant transfected cells showed that replication centers and MDC1 were located close to each other and they both were located near the nucleoli (Fig 8H). The localization of MDC1 in the periphery of NS1-labeled replication center foci was indistinct. Taken together, our data reveals that transfection with the NS2 splice donor mutant resulted in significant changes in the amounts and distributions of DDR proteins. The decreasing association of H2AX and MDC1 with the viral replication centers in cells transfected with the splice donor mutant (Fig 8E and 8H) is interesting as MVM studies have demonstrated a close localization between replication centers and DDR proteins [34]. These findings show that N-terminal sequence of NS2 is required for the normal progression of DNA damage response in infection, and the mutation of splice donor site has an effect on the recruitment of H2AX and MDC1 to the replication region where they most likely associate with NS1 in wt infection. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. Mutation of NS2 induce alteration in the amount and distribution of DDR proteins. Representative confocal images show the nuclear localization of NS1 (green), (A) γ-H2AX (magenta), (B) MDC1 (magenta) and DAPI staining (gray) in HeLa cells without transfection or at 24 hpt with wt, NS2 splice donor and acceptor mutants. Scale bars, 5 μm. (C) Total fluorescence intensities of γ-H2AX together with (D) its nuclear distribution as a function of increasing distance from the NE in nontransfected, wt and NS2 mutants transfected cells (n = 29). (E) Intensity line profiles of γ-H2AX (purple) and NS1 (green) measured through the nucleoli. (F) Fluorescent intensities and (G) nuclear localization of MDC1 from the NE (n = 28). The error bars show the standard error of the mean. (H) Intensity line profiles of MDC1 (purple) and NS1 (green) measured through the nucleoli. Statistical significances were determined using Dunnett’s multiple comparison test. The significance values shown are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). Fluorescent intensity profiles of (G) γ-H2AX (magenta) with NS1 (green), and (H) MDC1 (magenta) with NS1 (green) in zoomed areas. https://doi.org/10.1371/journal.ppat.1010353.g008

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010353

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.