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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles [1]

['Paulina Alatriste Gonzalez', 'Department Of Plant Pathology', 'University Of Kentucky', 'Lexington', 'Kentucky', 'United States Of America', 'Peter D. Nagy']

Date: 2022-08 

Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting numerous host components and rewiring cellular pathways to build extensive virus-induced replication organelles (VROs) in the cytosol of the infected cells. Most molecular resources are suboptimal in susceptible cells and therefore, tomato bushy stunt virus (TBSV) drives intensive remodeling and subversion of many cellular processes. The authors discovered that the nuclear centromeric CenH3 histone variant (Cse4p in yeast, CENP-A in humans) plays a major role in tombusvirus replication in plants and in the yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV indirectly alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation in situ within VROs by the co-opted glycolytic enzymes to support the energy requirement of virus replication. In summary, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication.

Biogenesis of viral replication organelles (VROs) containing membrane-bound viral replicase complexes is critical for replication of (+)RNA viruses. To achieve efficient VRO formation, RNA viruses reprogram host gene transcription through incompletely characterized processes. Using tombusviruses and the model host yeast, the authors discovered that the centromeric Histone 3 variant (CenH3 or CENP-A) is recruited into tombusvirus VROs. Tombusvirus replication protein and the viral RNA interact with CenH3 leading to partial sequestration of CenH3 into cytosolic VROs from the nucleus. The subversion of CenH3 leads to selective re-programming of gene expression of the host, resulting in increased production of glycolytic and fermentation enzymes. These enzymes are exploited by tombusviruses to produce ATP locally within the VROs to achieve robust replication.

Funding: This study was supported by the National Science Foundation (grant IOS-1922895) and National Institute of Food and Agriculture (2020-70410-32901) to P.D.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this paper, we studied the role of CenH3 in TBSV replication in yeast, plants and in vitro. Based on knockdown, mutation or over-expression experiments, we showed that CenH3/Cse4p acts as a cellular restriction factor against TBSV replication. CenH3/Cse4p was found to be partially re-targeted from the nucleus into the cytosolic VROs. In vitro works showed that CenH3/Cse4p binds to the viral RNA and acts as an RNA chaperone. Co-purification and pulldown experiments demonstrated interaction between CenH3/Cse4p and the viral p33 replication protein. However, subsequent analysis showed that TBSV hijacks CenH3/Cse4p into VROs to sequester this histone 3 variant away from the nucleus, which affects the expression of a set of host genes. These genes include pro-tombusviral host factors. We chose to further test the role of CenH3/Cse4p in regulating the glycolytic and fermentation pathways, which are co-opted by tombusviruses. These pathways are usurped by TBSV to provide plentiful ATP within VROs to fuel the activities of additional co-opted host proteins, such as Hsp70, the ESCRT-associated Vps4 AAA ATPase and DEAD-box helicases needed for robust viral replication [ 41 – 46 ]. Altogether, CenH3/Cse4p plays both pro-viral and restriction functions during tombusvirus replication.

The nucleus of a eukaryotic cell is full of nucleic acid binding proteins, which can potentially be used by the host to fight viral infections. Indeed, many well-characterized nuclear proteins are shuttling in and out of the nucleus, making possible that these cellular proteins could also function in the cytosol [ 16 , 25 – 28 ]. The histone H3 variant, CenH3 is essential for chromosome segregation by marking the centromere. This protein is so conserved in eukaryotes that the yeast Cse4p can complement the human CENP-A [ 29 ]. Nucleosomes containing CenH3 bind to long noncoding RNAs (called cenRNA) in the nucleus, which helps CenH3 to be localized to the centromeric portion of chromosomes [ 30 , 31 ]. However, CENP-A also localizes to noncentromeric regions (130 sites in yeast and 11,000 ectopic locations in the human chromosomes) in S phase [ 32 , 33 ]. These noncentromeric sites frequently represent intergenic and promoter regions with high histone turnover and transcriptional hotspots [ 33 – 36 ]. In yeast, high levels of Cse4p in promoter regions were related to down-regulation of gene expression [ 35 ]. Moreover, mislocalization and overexpression of CenH3 has been found in many cancers and associated with aneuploidy in Drosophila [ 37 – 40 ].

Using a library of temperature-sensitive mutants of yeast (a model host for TBSV), we identified Cse4 centromeric H3 protein variant as a restriction factor for TBSV replication [ 17 ]. Based on a protein network analysis [ 18 ], we found that Cse4 is one of the most highly connected nodes among the ~500 host factors identified, which affect TBSV replication, recombination or TBSV-host interaction in yeast [ 19 – 24 ]. This is a surprising discovery, because the DNA-binding nuclear histone proteins are not known to function as antiviral proteins against the cytosolic RNA viruses. Therefore, we decided to dissect the function of Cse4 and the plant CenH3 in TBSV replication.

TBSV codes for two viral replication proteins, termed p33 and p92 pol , which are essential for virus replication [ 13 , 14 ]. These replication proteins induce all the above cellular changes with major assistance from co-opted host enzymes and pathways [ 11 , 15 , 16 ]. Therefore, a major frontier in virus-host interaction studies is to advance our understanding how a (+)RNA virus can rewire cellular pathways and optimize the cellular milieu that then will support robust viral RNA replication. Yet, the picture of virus-host interactions is further complicated by host responses, including an arsenal of restriction factors, which inhibit the viral replication.

Positive-strand (+)RNA viruses use the abundant resources of the host cells to build large viral replication compartments/organelles (VROs), which support their replication in a membranous protective microenvironment [ 1 – 9 ]. Tomato bushy stunt virus (TBSV), a (+)RNA virus, is intensively studied to decipher virus-host interactions, virus replication and recombination. An emerging theme from TBSV studies is that this cytosolic replicating virus dramatically remodels subcellular membranes, hijacks various transport vesicles and co-opts numerous host proteins to facilitate various steps in the robust viral replication process [ 10 , 11 ]. Interestingly, however, the originally available resources in the uninfected host cells provide suboptimal conditions for robust TBSV replication. Accordingly, ever-increasing data show that TBSV rewires metabolic processes, alters the lipid compositions of the targeted endomembranes and organelles, and induces host gene expression to increase the abundance of host factors, which are co-opted for TBSV replication in the infected cells [ 10 – 12 ].

Results

The nuclear CenH3 histone variant restricts tombusvirus replication in yeast and plants To explore the possible role of Cse4p (CenH3) in tombusvirus replication, we used the temperature-sensitive haploid yeast strain with a mutated single copy of cse4-1 [17,47]. Partial inhibition of Cse4p by growing the yeast cse4-1 strain at the semi-permissive temperature (32°C) resulted in a ~4-fold increased level of TBSV replicon (rep)RNA [48,49] replication when compared with the BY4741 yeast strain carrying the WT copy of CSE4 (Fig 1A, compare lanes 13–16 to 9–12). TBSV replication was also higher in the cse4-1 strain than in the WT strain even at the permissive temperature (23°C, Fig 1A). This might indicate that a novel activity, not the canonical essential function of Cse4p in chromosome segregation, provides inhibitory effect against TBSV replication. Western blot analysis revealed that the tombusvirus p33 replication protein was expressed close to WT level in the cse4-1 strain (Fig 1A). We also tested the closely-related carnation Italian ringspot virus (CIRV), which replicates on the boundary membranes of mitochondria in contrast with the peroxisome-associated TBSV. CIRV replication is also increased by ~4-fold in cse4-1 strain at the semi-permissive temperature (Fig 1B). These findings suggest that Cse4p is a restriction factor for tombusvirus replication in different subcellular environments. PPT PowerPoint slide

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TIFF original image Download: Fig 1. The essential centromeric histone variant CenH3 is a restriction factor of tombusvirus replication in yeast and plants. (A and B) Top: Northern blot analyses show increased TBSV (A) or CIRV (B) repRNA accumulation in WT and cse4-1 yeasts grown at permissive and semi-permissive (32°C) temperatures. Middle: The accumulation level of repRNA was normalized based on 18S rRNA levels. Bottom: The accumulation levels of His 6 -p33 or the CIRV His 6 -p36 replication protein were measured by western blotting with anti-His antibody. (C) The effect of CenH3 overexpression on TBSV repRNA replication in yeast cells. Top: The 3’ end specific probe used for northern blot shows a reduction in the accumulation of TBSV repRNA in yeast expressing A. thaliana His 6 -tagged CenH3 (lanes 1–3) or S. cerevisiae Myc-tagged Cse4p (lanes 7–9) in comparison with the empty vector control (lanes 4–6). Bottom: Western blot analyses of the level of His 6 -p33, His 6 -AtCenH3 with anti-His antibody and Myc-Cse4 with anti-Myc antibody. (D) VIGS-based knockdown of CenH3 expression leads to increased level of TBSV replication in N. benthamiana plants. Top: Northern blot analysis of tombusvirus gRNA and sgRNA accumulation in CenH3-silenced plants inoculated with TBSV. VIGS was performed via agroinfiltration of tobacco rattle virus (TRV) vector carrying 5’ or 3’-terminal NbCenH3 sequences or 3’-terminal GFP sequences as control. Middle: Ethidium bromide-stained gel shows ribosomal RNA levels in each sample as a loading control. Bottom: CenH3-silencing restricts the growth of plants. NbCenH3 mRNA levels were analyzed by semi-quantitative RT-PCR in the silenced and control plants. Tubulin mRNA was used as a control. (E) Accumulation of CIRV gRNA and sgRNA in CenH3-silenced N. benthamiana plants was measured by Northern blot analysis. See further details in panel D. (F-H) Northern blot analyses of tombusvirus gRNA and sgRNA accumulation in N. benthamiana plants expressing CenH3 and inoculated with TBSV (F), CIRV (G) or CNV (H). Samples were taken 48 h (F), 72 h (G) or 84 h (H) after virus inoculation. Each experiment was performed at least three times. https://doi.org/10.1371/journal.ppat.1010653.g001 We used another approach to test the restriction function of CenH3 by expressing the WT Arabidopsis CenH3 in yeast replicating TBSV. We observed ~3-fold inhibition of TBSV repRNA accumulation in comparison with the control yeast (Fig 1C). Similarly, over-expression of the yeast Cse4p also inhibited TBSV repRNA replication in yeast (Fig 1C). These experiments confirmed that under these conditions, CenH3/Cse4 acts as a restriction factor during TBSV replication. To further explore if the plant CenH3 acts as a restriction factor of tombusvirus replication, we used a virus-induced gene silencing (VIGS) approach to deplete CenH3 level in Nicotiana benthamiana (Fig 1D, bottom panels) [50]. Replication of TBSV genomic (g)RNA was increased by ~3-to-5-fold in the CenH3 knockdown plants when compared to the non-silenced control plants two days after inoculation (Fig 1D, lanes 7–18 versus 1–6). Knockdown of CenH3 (TRV-5’CENH3, Fig 1D) rendered the plants smaller than the control (TRV-cGFP) plants, yet the knockdown plants supported higher levels of TBSV replication, suggesting that low CenH3 expression makes the plants more suited to support TBSV replication. Comparable experiments with CIRV showed that the CenH3 knockdown plants are indeed highly supportive of tombusvirus replication (Fig 1E). To further test the restriction function of CenH3 against tombusvirus replication in plants, we transiently expressed either NbCenH3 or AtCenH3 in N. benthamiana followed by inoculation of the same leaves with two peroxisome-associated tombusviruses (i.e. TBSV and the closely-related cucumber necrosis virus, CNV) and the mitochondrial membrane-associated CIRV. Northern blot analysis revealed ~8-to-10-fold reduction in TBSV, CNV and CIRV gRNA accumulation in the inoculated leaves (Fig 1F–1H). Therefore, all the above data support a strong tombusvirus restriction function of the plant CenH3 histone variant.

Recruitment of the nuclear CenH3 histone variant into the tombusvirus replication organelles in plants To test if the restriction function of CenH3 is performed in the nucleus or in the cytosol, where tombusviruses assemble the large viral replication organelles (VROs), we co-expressed TBSV p33-BFP replication protein and the GFP-tagged Arabidopsis CenH3, the ortholog of the yeast Cse4, in transgenic N. benthamiana leaves expressing the RFP-H2B (histone H2B) nuclear marker protein (Fig 2A). Confocal laser microscopy analysis revealed the co-localization of p33-BFP and GFP-CenH3 in N. benthamiana cells replicating CNV (Fig 2A). Interestingly, a portion of GFP-CenH3 was still localized in the nucleus marked by the RFP-H2B marker protein in plant cells infected with CNV (Fig 2A). In contrast, GFP-CenH3 was exclusively localized to the nucleus in mock-inoculated plant leaves under these transient expression conditions (Fig 2A, top image). Importantly, the re-localized GFP-CenH3 in the cytosol was present in the TBSV VROs marked by both p33-BFP and RFP-SKL peroxisome luminar marker protein (Fig 2B). The expression of p33-BFP alone (in the absence of TBSV infection) facilitated the partial re-localization of GFP-CenH3 into VRO-like structures (Fig 2B), albeit this process was not as robust as in the case of TBSV or CNV infections. We also performed comparable experiments with the mitochondrial CIRV in either transgenic RFP-H2B or WT N. benthamiana plants. The results showed partial re-localization of GFP-CenH3 into CIRV-induced VRO structures marked by p36-BFP and RFP-CoxIV mitochondrial marker protein (Fig 2C). The expression of p36-BFP alone (in the absence of CIRV infection) did not induce the re-localization of GFP-CenH3 into VRO-like structures (Fig 2C). Based on these results, we suggest that tombusvirus infections of N. benthamiana plants induce the partial re-localization of the nuclear GFP-CenH3 into the cytosolic VROs. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Re-distribution of nuclear CenH3 to the cytosolic sites of viral replication in plants. Confocal laser microscopy images show the localization of GFP-CenH3 in N. benthamiana cells. (A) First panel: In the absence of virus replication, GFP-CenH3 localizes solely in the nucleus, as shown by its co-localization with the histone RFP-H2B. Second panel: Co-localization of p33-BFP replication protein and GFP-CenH3 in cells replicating CNV. The VROs are marked with white arrows. Third and fourth panels: The re-distributed GFP-CenH3 is present in the VROs, marked by p33-BFP and RFP-SKL peroxisomal marker protein. Note that a portion of GFP-CenH3 remains in the nucleus. (B) GFP-CenH3 partially re-localizes into the VRO-like structures, in the absence of TBSV replication when only TBSV p33-BFP is expressed. (C) First panel: Co-localization of CIRV p36-BFP replication protein and GFP-CenH3 in cells replicating CIRV. Second and third panels: GFP-CenH3 is re-distributed into the CIRV-induced VROs consisting of aggregated mitochondrial membranes, marked with both p36-BFP and RFP-CoxIV. Fourth panel: GFP-CenH3 does not re-localize into the VRO-like structures, in the absence of CIRV replication when only p36-BFP replication protein is expressed. https://doi.org/10.1371/journal.ppat.1010653.g002 We also performed subcellular localization experiments in WT yeast cells expressing YFP-Cse4 and co-expressing CFP-p33 together with p92pol and the repRNA to induce VRO formation [51]. Time point experiments revealed the partial co-localization of YFP-Cse4 with CFP-p33 at 12 h time point after induction of protein expression (S1 Fig). The co-localization of YFP-Cse4 and CFP-p33 was even more pronounced at the 16 h and 24 h time points (S1 Fig). This is in contrast with the nuclear localization of YFP-Cse4 in WT yeast in the absence of viral components (S1 Fig). Based on these data, we propose that Cse4p is partially re-localized from the nucleus into the cytosolic VROs marked by p33 replication protein in yeast. Altogether, CenH3 and the orthologous Cse4p are re-targeted by TBSV in plant and yeast cells.

Altering host gene expression by TBSV depends on CenH3 in yeast and plants Previous work with cse4-1 yeast suggested that Cse4p has noncanonical functions in yeast outside of the centromeric area of the chromosome [33,34]. Cse4p acts as a noncanonical regulator of selected number of host genes via replacing histone molecules on the intergenic and promoter regions in chromosomal DNA [33,34]. Interestingly, genes whose expression is affected by Cse4p include several pro-viral host factors needed for robust TBSV replication. These host genes include several glycolytic and ethanol-producing enzymes [33,34], which are also selectively hijacked by TBSV into VROs to provide plentiful ATP locally to promote efficient TBSV replication [59–61]. Indeed, we have confirmed that mRNA expression for pyruvate kinase (PK, termed Cdc19 in yeast), Eno2 (Enolase 2), Pgk1 (phosphoglycerate kinase) and Pdc1 (pyruvate decarboxylase) glycolytic and fermentation enzymes and the pro-viral Ded1 DEAD-box helicase was increased in cse4-1 yeast at the semi-permissive temperature (Fig 4A). On the contrary, the expression of SSA1 (Hsp70) and TEF1 (eEF1A) genes has not changed in cse4-1 yeast at the semi-permissive temperature (Fig 4A). Ssa1 and Tef1 are key co-opted host proteins during TBSV replication in yeast [22,45,62,63]. The latter findings suggest that expression of a group of, but not all the pro-viral genes is affected by Cse4p in yeast. PPT PowerPoint slide

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TIFF original image Download: Fig 4. TBSV reprograms host gene expression via co-opting CenH3 in yeasts and plants. (A) Upregulation of mRNA expression of pro-viral host factors in cse4-1 mutant yeast. The mRNAs levels for glycolytic enzymes and other host proteins shown were estimated by semi-quantitative RT-PCR in total RNA samples obtained from WT or cse4-1 yeast cells grown at 32°C for 12 h. (B) Upregulation of mRNA expression of pro-viral host factors in CenH3-silenced N. benthamiana plants. The mRNA levels were estimated by semi-quantitative RT-PCR in total RNA samples obtained from either CenH3 knockdown or control plants (TRV-cGFP), 12 d after VIGS treatment. (C) Expression levels of mRNAs of N. benthamiana glycolytic and fermentation enzymes were estimated by real-time qRT-PCR in total RNA samples obtained from CenH3 knockdown (KD) or control plants (TRV-cGFP) in the absence or presence of TBSV replication. The mRNA level in TRV-cGFP control plants is chosen as 1 unit for each gene tested. (D) Expression levels of mRNAs of N. benthamiana glycolytic and fermentation enzymes were estimated by real-time qRT-PCR in total RNA samples obtained from either mock or TBSV inoculated plants, 2 (for inoculated leaves) or 4 (for systemic leaves) days post inoculation. (E) mRNAs expression levels of N. benthamiana hypoxia-related transcription factors were estimated by real-time qRT-PCR in the same total RNA samples as in panel C. Each experiment was repeated three times or more. https://doi.org/10.1371/journal.ppat.1010653.g004 VIGS-based knockdown of CenH3 level in N. benthamiana also led to enhanced expression of Eno2, Pgk1 and Pdc1 glycolytic/fermentation enzymes and the pro-viral RH20 (ortholog of the yeast Ded1) DEAD-box helicase (Fig 4B). Moreover, we have found that CenH3 knockdown in combination with TBSV infection of N. benthamiana led to the highest expression levels of PK1, Pgk1, GAPC1 (glyceraldehyde-3-phosphate dehydrogenase) and Pdc1 (Fig 4C). TBSV infection did also enhance the expression level of PK1, Pgk1, GAPC1 and Pdc1 by ~4-to-8-fold (Fig 4D). CenH3 expression was increased by ~3-fold at 2 dpi, followed by close to normal level of CenH3 expression at 4 dpi (Fig 4D). On the contrary, the expression of pro-viral Hsp70-1 and eEF1A plant genes [22,45,62,63] did not change in CenH3 knockdown plants (Fig 4B). Thus similar to the observations in yeast, the latter findings suggest that expression of a selective group of, but not all the pro-viral genes is affected by CenH3 in plants. Because the fermentation enzymes (Adh1 and Pdc1) are induced during hypoxia (low O 2 level due to plant submersion in water) by ERF-VII transcription factors in Arabidopsis plants, we measured the expression levels of RAP2.12, HRE1 and HRE2, which are known hypoxia transcription factors [64–66]. We have found that CenH3 knockdown in combination with TBSV infection of N. benthamiana led to the highest expression levels of RAP2.12, HRE1 and HRE2, which showed ~4-to-9-fold increase (Fig 4E). Interestingly, CenH3 knockdown or, separately, TBSV infection also enhanced the expression level of RAP2.12, HRE1 and HRE2 (Fig 4E). These surprising findings on the shared function of TBSV infection and CenH3-based regulation of expression of glycolytic/fermentation enzymes and hypoxia-related transcription factors led to a new working model. The emerging idea is that TBSV hijacks CenH3/Cse4 from the nucleus into the VROs to affect the normal cellular gene-regulatory function of this conserved histone variant. This leads to alteration of gene expression of selected group of host genes whose expression is affected (directly or indirectly) by the noncanonical function of CenH3/Cse4.

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