Zika Virus induced mitotic arrest, apoptosis, DNA damage response and autophagy: recipe for disaster?

Zika Virus (ZIKV) was first isolated from monkeys in 1947 and until 2007 only isolated cases of infection in humans were reported although serologic studies suggested widespread distribution in Africa and Southeast Asia. In 2007 however ZIKV of the Asian lineage caused an outbreak in Yap/Federal Micronesia followed by an outbreak in French Polynesia in 2013 and the current outbreak in Central & South America and the Caribbean. ZIKV is mainly transmitted via infected Aedes spp.mosquitoes but sexual transmission via semen of infected man has also been reported in addition to contaminated blood and  blood products. 

Clinical symptoms caused by ZIKV infection are very similar to that of other arboviral infections such as Dengue Virus (DENV) or Chikungunya Virus (CHIKV) which often co-circulate in areas affected by ZIKV and more often than not ZIKV infection is asymptomatic. In general, ZIKV may cause fever, maculopapular rash, and arthralgia or conjunctivitis. Following the outbreak in French Polynesia 2013 -2014 an increase in Guillain-Barré Syndrome (GBS) has been observed (albeit only by retrospective analysis) and -based on data obtained during the current outbreak-a link between the onset of microcephaly and ZIKV infection during the early stages of gestation has been proposed (but not verified).

Experimentally, the neurotrophic properties of ZIKV have been demonstrated in mice injected with mouse adapted strain of ZIKV intraperitoneally and a isolates directly of obtained from monkeys that were injected intracelebral. In both cases, virus replicated in neuronal cells of the CNS and the brain with extensive apoptosis of infected cells. A subtraction of infected mice also showed paralysis or morbidity, suggesting that in mice ZIKV can induce a severe infection. Cerebrospinal fluid from GBS patients has been shown to induce neuronal apoptosis suggesting the presence of neurotic factors, but at present this has not been confirmed to be the case for ZIKV induced GBS and the relevance to disease pathology is unclear. In the case of ZIKV induced microcephaly, ZIKV may be transmitted to the embryo and/or foetus and interfering with brain development via the placenta. Indeed, ZIKV has been isolated in the placenta of ZIKV positive pregnant women, suggesting that ZIKV might be transmitted via exosomes (similiar to HCV) that infect embryonic and/or foetal cells of the neuroepithelium or directly infects the cerebral cortex during the earliest stages of brain development. Alternatively, ZIKV infection of the placenta disrupts the outer layer of the placenta thus inducing either miscarriage or -in the absence of viral induced miscarriage- contribute to viral induced microcephaly by disrupting placental signals to the developing brain and inducing an inflammatory response with the developing foetus (similar  to MHV-68). In any case, ZIKV has been detected both in the amniotic fluid of at least two pregnant women whose foetuses were diagnosed with microcephaly as well as in foetal brain tissue, suggesting that the currently circulating ZIKV strains are indeed neurotrophic and might contribute to the onset of microcephaly. 

Experimentally, younger mice infected with ZIKV derived from monkey exhibit more severe brain damage compared to older mice suggesting that ZIKV indeed might indeed interfere with the developing brain. Human induced pluripotent stem cells differentiated into forebrain specific human neural progenitor cells (hNPC) that have been infected with the original ZIKV strain MR766 used in the mice studies published in 1952 , suggest that ZIKV induces cell cycle arrest and apoptosis (and subsequent growth arrest) 72 hrs p.i. . 

Gene expression analysis of ZIKV infected hNPC showed that ZIKV infection alters the expression of at least 7000 genes related to cell cycle regulation, the DNA damage response, autophagy, apoptosis and other cellular processes suggesting that ZIKV might indeed interfere with these pathways as discussed before and as detailed below. In general, ZIKV infection downregulates the expression of genes encoding for proteins regulating DNA repair and cell cycle progression whilst upregulating the expression of genes ending for proteins regulating autophagy and the ER stress response, suggesting that ZIKV induces the formation of stalled replication forks, inhibiting the progression of S to G2 phase, inducing mitotic arrest whilst promoting the formation of lipid droplets and autophagosome-like vesicles thus supporting viral replication and dissemination. 

ZIKV: inhibition of DDR, DNA replication and the cell cycle 

As discussed before, both retroviruses and positive strand RNA viruses (in addition to DNA viruses interfere with the DNA damage response which can be induced by the expression of viral proteins, endogenous reactive oxygen species or by exogenous agents including UV and chemotherapeutic drugs. Depending on the nature of the DNA damage, different repair pathways can be induced, with the homologous DNA repair pathway (HR) predominant in S and G2 cells and the Non-Homologous End Joining (NHEJ; which itself can be distinguished between a conservative and alternative pathway) present in all phases of the cell cycle, (probably) including M phase. Upon successful DNA repair, cells reenter the cell cycle whereas incomplete DNA repair can induce an arrest in S phase and subsequent G2 arrest; failure to activate the G2/M checkpoint may result in mitotic arrest followed by apoptosis or abberant mitosis, thus contributing to the development of tumour cells. 

Figure: General outline of the connection between the DNA damage response, cell cycle regulation and
autophagy

In the case of positive RNA viruses, the expression of IBV and SARS nsp13 protein has been shown to induce stalled replication forks and the expression of the N protein derived from different Coronaviruses has been shown to delay the progression from S to G2 probably by inducing ATR (and thus potentially the ATR dependent DNA damage response). Delaying the onset of G2 might favour viral replication not only by extending G2 and thus providing enough time to replicate but also provide cellular resources such as enzymes or nucleotides required for viral replication. Failure to resolve DNA damage that naturally occurs during DNA replication or persistent activation of the DDR by viral proteins however might contribute also to mitotic arrest and thus induce apoptosis.

Table: Genes that are down-or unregulated in ZIKV infected cells: DDR

In the case of ZIKV infected hNPC, viral infection decreases the expression of genes encoding proteins that are required for at least three distinct pathways, namely HR dependent pathways induced by both ATM and ATR, as well as the POLQ dependent Base-Excision Repair (BER) pathway and the Fanconi Anemia (FA) pathway that intersects with the ATR pathway. In contrast to the decreasing the expression of genes related to the main DNA damage repair pathways, the expression of at least one gene, STKL18, related to the Nucleotide Excision Repair and a NBS1 independent repair pathway, ATMIN, is increased following ZIKV infection. The relative contribution of these pathways to DNA repair in general are however minor and if these pathways are functional in ZIKV infected cells is not known presently. 

Figure: ZIKV potentially inhibits the DDR at multiple sites whilst inducing the acetylation of NBS1 and
the initial recruitment of sensors to sites of DNA damage induced by stalled DNA replication

Similar to genes encoding proteins regulating the DDR, the expression of genes encoding proteins regulating the cell cycle, in particular genes related to the progression of G1, G1 to S, S to G2 and those related to the control of mitotic progression and chromosome segregation, are generally downregulated with the notable exception of NEDD1. Since NEDD1 encodes for a protein involved in the duplication of the centriole and spindle assembly, it might be possible that the over expression of NEDD-1 induced by ZIKV infection might induce mitotic arrest due to the presence of multiple centrioles. In addition to genes encoding for proteins such as Cyclin B1, CDK2, or CDC25A, ZIKV expression also downregulates the expression of genes related to DNA replication, suggesting that ZIKV infection might also induce the formation of stalled replication forks, which could be measured experimentally using a CldU/IdU based assay.

Consequently, it can be assumed that ZIKV infection induces the arrest of infected cells in G2 and/or M phase of the cell cycle -which is supported by flow cytometry based cell cycle analysis of infected hNPC cells. Using Histone H3-P(Ser-10) as a marker for mitotic cells it should be easy to discriminate cells arrested in G2 from cells arrested in M phase. Confocal microscopy can be used to further distinguish cells arrested in prophase from those in metaphase or anaphase. Based on the data obtained from analysing the transcriptome, it might be however possible that ZIKV arrests cells in multiple stages of mitosis, since not only the formation of spindle poles might be affected but also the progression of anaphase and thus the onset of telophase. 

Table: Genes down- or upregulated in ZIKV infected cells: cell cycle

Figure: Cell Cycle arrest in ZIKV infected cells 

ZIKV: promotion of autophagosome formation, ER stress response and lipid droplet formation

In contrast to genes related to the control of the cell cycle, the DDR and DNA replication, ZIKV infection generally induces the expression of genes encoding for proteins required for the initiation of autophagosome formation including those encoding for proteins that increase the pool of cytosolic LC3 (SIRT1 and ATG4A) or induce the formation of autophagosomes (STK38L, ATG16L1, RABGAP1 and ULK1) whereas selective (p62/SQSTM-1 dependent) autophagy is inhibited maybe due to decreasing the expression of KLF4 and stress induced autophagy by decreasing the expression of Caspase-2. The maturation of the autophagosome and/or lysosome however might be inhibited by decreasing the expression of LAMP2, similar to the formation of antiviral autophagic clusters by decreasing the expression of ISG15. 

Table: Genes are down-orupregualted in ZIKV infected cells: Autophagy and UPR

Figure: ZIKV inhibits selective autophagy whilst promoting the formation of viral RC via increased autophagosome/EDEMosome formation

Increasing the expression of Optineurin and BNIP-3 might promote the formation of mitophagsomes and thus lipophagy as described before. The synthesis of lipid droplets at the ER might be aided by increased expression of DCP2 and XRN1, and similar to cells infected with HCV, ZIKV might promote the formation of ER localised lipid droplets by decreasing the number of P-bodies whilst decreasing the formation of stress granules. 

Figure: Lipid droplets are degraded via ZIKV induced mitophagy

The formation of lipid droplets and autophagosomes in ZIKV infected cells might also be supported by increasing the expression of proteins regulating the ER stress response/Unfolded Protein Response (UPR) such as SEC16A (inducing the formation of COPII vesicles) and EDEM1 (inducing EDEMosomes). In the case of BNIP-3, increased BNIP-3 might either induce mitophagy or induce the release of Ca2+ from the ER and thus promote mitochondrial apoptosis; alternatively BNIP-3 might induce the depolarisation whilst the Optineurin facilitates the clearance of damaged mitochondria by mitophagy. 

The induction go both the PERK and IRE-1 induced ER stress however might be inhibited, thus preventing -at least partially- the induction of UPR induced apoptosis. In contrast, only the ATF mediated ER stress response might be still active, resulting in delayed or decreased expression of CHOP. 

Figure: ZIKV and the ER stress response

In summary, ZIKV might induce the formation of autophagosomes or autophagosome-like structures by inducing the expression of proteins that are known inducers of autophagosome formation as well as inducing at least a partial ER stress response whilst inhibit selective autophagy. The formation of autophagosomes and the inhibition of p62/SQSTM-1 dependent selective autophagy has been confirmed in skin fibroblasts and keratinocytes whilst the induction of the UPR by ZIKV has not been demonstrated yet. In addition to inducing autophagosomes, the formation of antiviral clusters might be inhibited,similar to neuronal cells infected with Yellow Fever Virus, probably due to the downregulation of both ISG15 and p62/SQSTM-1 expression. Propagation of lipid droplet formation and lipophagy by decreasing the number of P bodies and promoting the formation of mitophagosomes fused with LD's might promote viral replication by providing free fatty acids but probably not by providing a scaffold for the viral replication complex (in contrast to HCV).  If the upregulation of SEC16A contributes to the release of exsomes containing viral particles similar to HCV remains also to be seen. 

Figure: ZIKV induced pathways are linked, controlling cell proliferation in multiple
ways

In conclusion, ZIKV induces apoptosis by regulating by generally downregulating the expression of genes encoding proteins that regulate the replication of cellular DNA, the DNA damage response and the cell cycle as well as upregulating genes expressing proteins that are proapoptotic (DIABLO and FADD). Apoptosis is therefore induced by an arrest in G2 and M phase of the cell cycle as well as promoting the depolarisation of mitochondria. Inducing the expression of genes that promote autophagosome formation as well as formation and degradation of lipid droplets in contrast promotes viral replication.The spatial expression pattern of these genes however remains to be elucidated, but in general the analysis of the transcriptome on ZIKV infected hNPC cells provides an insight in the complex interaction of the various pathways regulated by ZIKV and ZIKV related viruses. 

Further reading

DICK GW, KITCHEN SF, & HADDOW AJ (1952). Zika virus. I. Isolations and serological specificity. Transactions of the Royal Society of Tropical Medicine and Hygiene, 46 (5), 509-20 PMID: 12995440 

DICK GW (1952). Zika virus. II. Pathogenicity and physical properties. Transactions of the Royal Society of Tropical Medicine and Hygiene, 46 (5), 521-34 PMID: 12995441 

  

Calvet, G., Aguiar, R., Melo, A., Sampaio, S., de Filippis, I., Fabri, A., Araujo, E., de Sequeira, P., de Mendonça, M., de Oliveira, L., Tschoeke, D., Schrago, C., Thompson, F., Brasil, P., dos Santos, F., Nogueira, R., Tanuri, A., & de Filippis, A. (2016). Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study The Lancet Infectious Diseases DOI: 10.1016/S1473-3099(16)00095-5 

  

Brasil, P., Pereira, Jr., J., Raja Gabaglia, C., Damasceno, L., Wakimoto, M., Ribeiro Nogueira, R., Carvalho de Sequeira, P., Machado Siqueira, A., Abreu de Carvalho, L., Cotrim da Cunha, D., Calvet, G., Neves, E., Moreira, M., Rodrigues Baião, A., Nassar de Carvalho, P., Janzen, C., Valderramos, S., Cherry, J., Bispo de Filippis, A., & Nielsen-Saines, K. (2016). Zika Virus Infection in Pregnant Women in Rio de Janeiro — Preliminary Report New England Journal of Medicine DOI: 10.1056/NEJMoa1602412 

Chan JF, Choi GK, Yip CC, Cheng VC, & Yuen KY (2016). Zika fever and congenital Zika syndrome: An unexpected emerging arboviral disease? The Journal of infection PMID: 26940504 Adibi, J., Marques, E., Cartus, A., & Beigi, R. (2016). Teratogenic effects of the Zika virus and the role of the placenta The Lancet DOI: 10.1016/S0140-6736(16)00650-4 

Bell TM, Field EJ, & Narang HK (1971). Zika virus infection of the central nervous system of mice. Archiv fur die gesamte Virusforschung, 35 (2), 183-93 PMID: 5002906 

Tang, H., Hammack, C., Ogden, S., Wen, Z., Qian, X., Li, Y., Yao, B., Shin, J., Zhang, F., Lee, E., Christian, K., Didier, R., Jin, P., Song, H., & Ming, G. (2016). Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth Cell Stem Cell DOI: 10.1016/j.stem.2016.02.016

Biegel JM, & Pager CT (2016). Hepatitis C Virus Exploitation of Processing Bodies. Journal of virology PMID: 26937026 

Katzenell S, & Leib DA (2016). Herpes simplex virus and interferon signaling induce novel autophagic clusters in sensory neurons. Journal of virology PMID: 26912623 

Ryan EL, Hollingworth R, & Grand RJ (2016). Activation of the DNA Damage Response by RNA Viruses. Biomolecules, 6 (1) PMID: 26751489 

Riz I, Hawley TS, & Hawley RG (2015). KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models. Oncotarget, 6 (17), 14814-31 PMID: 26109433 

Park JM, Choi JY, Yi JM, Chung JW, Leem SH, Koh SS, & Kang TH (2015). NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair. Biochemical and biophysical research communications, 461 (3), 543-8 PMID: 25912875 J

Joffre C, Codogno P, Fanto M, Hergovich A, & Camonis J (2016). STK38 at the crossroad between autophagy and apoptosis. Autophagy, 1-2 PMID: 26890257 

Joffre C, Dupont N, Hoa L, Gomez V, Pardo R, Gonçalves-Pimentel C, Achard P, Bettoun A, Meunier B, Bauvy C, Cascone I, Codogno P, Fanto M, Hergovich A, & Camonis J (2015). The Pro-apoptotic STK38 Kinase Is a New Beclin1 Partner Positively Regulating Autophagy. Current biology : CB, 25 (19), 2479-92 PMID: 26387716 

Kanu N, & Behrens A (2007). ATMIN defines an NBS1-independent pathway of ATM signalling. The EMBO journal, 26 (12), 2933-41 PMID: 17525732 Matsui A, Kamada Y, & Matsuura A (2013). The role of autophagy in genome stability through suppression of abnormal mitosis under starvation. PLoS genetics, 9 (1) PMID: 23382696 

Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, Perera-Lecoin M, Surasombatpattana P, Talignani L, Thomas F, Cao-Lormeau VM, Choumet V, Briant L, Desprès P, Amara A, Yssel H, & Missé D (2015). Biology of Zika Virus Infection in Human Skin Cells. Journal of virology, 89 (17), 8880-96 PMID: 26085147 

Beller M, Thiel K, Thul PJ, & Jäckle H (2010). Lipid droplets: a dynamic organelle moves into focus. FEBS letters, 584 (11), 2176-82 PMID: 20303960 

Bukong TN, Momen-Heravi F, Kodys K, Bala S, & Szabo G (2014). Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS pathogens, 10 (10) PMID: 25275643 

Shrivastava S, Devhare P, Sujijantarat N, Steele R, Kwon YC, Ray R, & Ray RB (2015). Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway. Journal of virology, 90 (3), 1387-96 PMID: 26581990 

Schorey JS, Cheng Y, Singh PP, & Smith VL (2015). Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO reports, 16 (1), 24-43 PMID: 25488940

Positive strand RNA viruses and the DDR: cell cycle arrest and autophagy

Both DNA and RNA viruses are dependent of cell proliferation for replication. Thus viruses often posses proteins which are able to interact with cellular proteins which are responsible for maintaining the regulation of the cell cycle. In general viruses could either slow the cell cycle or activate the cell cycle.

Figure: Viruses and the DDR: multiple points of action

In the case of HIV-1, the viral R protein (VpR ) was identified to be responsible for accumulation of  G2 arrested cells. by preventing the activation of  the cyclinB/CDK 1 through the inhibition of a phosphatase, cdc25A, and the activation of a kinase, Wee1. Both enzymes are hypophosphorylated in HIV-1 infected cells and VpR may act through the activation of protein phosphatase 2A (PP2A)

and thus blocks the G2-M transition. In this context it is very interesting to note that VpR -similar to coronaviral N protein is also located in the nucleolus of the cell. Another example is the paramyxovirus Simian parainfluenza virus 5, which is able to slow the progression both G1 into S and G2 into M but not of a G1 or G2 arrest .The V protein of Simian Virus 5 binds to a protein involved in DNA repair, DDB1 (DNA damage binding protein). DDB1 becomes activated upon binding through V. Activation of  DDB1 in turn leads to slowing the progression of G2 into M and binds to the Retinoblastoma protein and thus prevents entering of the S phase through non-activation of E2F. Because of the delayed activation of E2F, the transactivation of the genes necessary for successful entry into the S phase is also delayed.

Coronavirus and the cell cycle: Induction of p53 by ATR induces S phase arrest

The expression of the nucleocapsid (N) protein of Infectious Bronchitis Virus (IBV) or Severe Acute Respiratory Syndrome Virus (SARS) in Vero cells decreases not only cell proliferation in the absence of apoptosis but also arrests the cell cycle at S phase as well as inducing in a significant number of cells a mitotic defect as indicated by the presence of a cleavage furrow.  

Figure: Localisation of Coronavirus N protein and mitotic arrest

Indeed, bivariate flow cytometry of BrdU incorporation and Propidium iodide content and subsequent dot plot analysis by gating for G0/G1, S and G2/M phase showed that the number of cells in S phase increased by 20% if the cells were transfected with IBV N compared to mock transfected or cells transfected with empty vector as well as an increase in cells with DNA content higher than 4n,indicating not only that the expression of IBV N delays the cell cycle but also might induce mitotic defects. Similar the cell cycle is delayed in Vero cells arrested in G2/M with Nocadazole and released for 24 hrs by replacing the Nocadazole containing medium with fresh DMEM, indicating that the expression of N in Vero cells does delay the progression of Vero cells from S phase into G2 phase of the cell cycle. In Vero cells infected with a Vero adapted IBV Beaudette US strain a similar delay can also be observed (albeit to a lower extent) indicating that viral replication does not antagonise the ability of N to delay the cell cycle. In contrast to Vero cells, the expression of IBV N in Saos-2 cells that are deficient for p53 does not induce a delay or arrest in the cell cycle indicating that p53 is required for inducing the observed delay in cell cycle progression.

Figure: Expression of IBV N in Vero cells but not in Saos-2 cells induces cell cycle arrest
that it can be abrogated by Caffeine

p53 can be activated by ATR as a result of nucleolar stress and since the expression of the expression of the coronaviral N protein has been proposed to induce nucleolar stress, treatment of cells transfected with SARS or IBV N with an inhibitor of ATR should therefore reverse the activation of p53 by ATR. Indeed, Vero cells transfected with either IBV or SARS N protein and treated with 4 mM Caffeine do not exhibit an increase in S phase  -indicating that N protein induced S phase arrest is indeed mediated by an activation of ATR- but instead exhibit an increase in G0/G1 phase of the cell cycle indicating that N inhibits the progression of cells from G1 into S phase. Indeed, the expression of N does not only induce nucleolar stress but also inhibits Cyclin D1 as well as Cyclin A and E thus inhibiting entry into S phase in cells treated with Caffeine.

Figure: The expression of SARS N in A549 cells induces a transient
arrest in S phase

In addition, Caffeine treatment of Vero cells transfected with IBV N also increases the percentage of cells in G2/M indicating the activation of the G2/M checkpoint and flow cytometry analysis of Caffeine treated Vero cells transfected with SARS N confirms that the expression of SARS N induces a G2 rather than a mitotic arrest since SARS N does not increase Histone H3(Ser-10) phosphorylation.

In conclusion, the expression of IBV and SARS N induces an arrest primary in S phase of the cell cycle that is at least partially dependent on the induction of p53 by ATR. Abrogation of ATR dependent cell cycle arrest induces an arrest in late G1/early S phase and an arrest in G2 -but not M- phase.

Figure: The expression of SARS N in Vero cells induces p53 (A), p21 (B), CHK-1 (C) and but not mitotic arrest (D)

As discussed before, the expression of N might induce DRAM-1 and thus autophagy as a result of nucleolar stress that might promote viral replication by promoting the formation of viral replication centres.  In budding yeast, ATR mediated induction of autophagy has been linked to the induction of anaphase and recent evidence suggests that LC3 positive structures -probably mitophagosomes- are present throughout mitosis. Induction of autophagy by N therefore might be responsible for a premature onset of anaphase and thus inducing the formation of the midbody. Since aberrant cytokinesis is only present in a subset of cells expressing N, the majority of cells might not undergo premature mitosis but instead, the induction of autophagy might be a contributing factor to N induced S-G2/M arrest since autophagy can arrest cells by increasing levels of phosphorylated Cdc2(Y15), Cyclin A, E and B1, and Rb (S807/811) and decreasing Cyclin D1. Furthermore, Coronavirus N induced autophagy might also attenuate the DNA damage response by degrading Chk-1 via chaperone-mediated autophagy.

In addition to N, the expression of IBV and SARS nsp-13 has been shown to induce the DNA damage response as well as inducing S phase arrest via ATR. In this case, ATR is activated by stalled replication forks due to the inhibition of  p125 subunit of DNA polymerase δ and -similar to IBV and SARS N- the activation of p53 might induce autophagy. Since nsp-13 localises both to the nucleus and the cytoplasm, nsp-13 might trigger distinct events at different times of the viral replication cycle. ER localised nsp-13 might trigger the ER stress response and akin to (and maybe in addition to) nsp-4 and -6, promote the formation of viral RC by initiating the formation of omegasome-like structures. Nuclear nsp-13 in contrast inhibits DNA replication and induces the ATR response which in turn might facilitate the formation of RC indirectly by inducing the formation of autophagosomes and/or by inducing a S-G2/M arrest thus preventing degradation of the viral genome during mitosis as well as extending the cell cycle to allow viral replication. The individual contributions of both N and nsp-13 are however not known and it remains to be seen if the localisation of nsp-13 is altered during viral replication, i.e. if nsp-13 undergoes nuclear-cytoplasmic shuttling. It should also be noted that the expression of SARS N induces the activation of caspase-3 in the absence of depolarised mitochondria and in the absence of apoptosis. N and/or nsp-13 induced autophagy therefore might also promote the degradation of proapoptotic proteins or promote mitophagy and thus prevent Coronavirus induced apoptosis in addition to promoting the formation of viral RC. Interestingly, activated Caspase-3 localises to the nucleolus in A549 cells transfected with SARS N without -as indicated by staining of SARS N transfected cells with HSP-60 and Bcl-2 as well as flow cytometry detection of activated Bax- the presence of damaged mitochondria.

Figure: SARS N induces the localisation of active Caspase-3 to the nucleolus

Figure: Activity of Caspase-3 is not impaired in A549 cells transfected
with SARS N

Figure: SARS N does not impair mitochondrial integrity in A549 cells

In human MRC-7 cells the disintegration of the nucleolus has been shown to precede cell cycle arrest and p53 activation, suggesting that the nucleolar localisation of active Caspase-3 might contribute to nucleolar stress induced by the expression of SARS-CoV N protein, but further studies are needed to determine the pathway of caspase-3 activation and if damaged mitochondria are degraded by mitophagy. Also, it has to be determined of the induction of ATR by N or any other coronaviral protein does prevent the activation of caspase-3 dependent apoptosis. Alternatively, the expression of nsp-13 or N might induce the ER stress response and thus protects cells from caspase-3 dependent apoptosis.

In conclusion, both the expression of N and nsp-13 induces the ATR dependent DNA damage response as well might prevent the execution of the DDR by abrogating downstream signaling pathways thus arresting cells in S and/or G2 phase of the cell cycle. In the case of N, inhibition of G1 and S phase cyclins, namely Cyclin D1, A, and E, contributes to the observed arrest. The induction of p53 via ATR by N or nsp-13 might however contribute to the induction of Interferon type I signaling, which can be counteracted by the expression of the  PLP2 catalytic domain of nsp-3 . PLP2 therefore might not only inhibit antiviral singling but also partially prevent N induced cell cycle arrest, namely pathways induced by p53 such as the degradation of CHK-1 and the increase of Cyclin E m A, and B as well as the decrease of Cyclin D1, thus preventing S phase arrest in addition to prevent mitotic defects. So far however this has not been experimentally been proven.

Figure: IBV N, nsp-13, and nsp-14: induction of ATR and ATM dependent pathways

Furthermore, it needs to be determined why the expression of the TGEV N protein in contrast to the N protein derived from IBV, SARS, or MHV does induce p53 dependent apoptosis. Similar to SARS N, TGEV N induces a cell cycle arrest (in S and G2/M) and the activation of Caspase-3; unlike in A549 cells expressing SARS N however, Bax translocates to the mitochondria and induces the depolarisation of the mitochondrial membrane. It might be possible that in PK-15 autophagy is impaired and damaged mitochondria might accumulate. One possibility is that both SARS and IBV N do increase the expression of both Bak and Mcl-1 whereas TGEV N does not.

Finally, it needs to be determined if the nucleolar localisation of other viral proteins such as the core protein of JEV induces the activation of ATR as a result of nucleolar stress.

Further reading

Flynn RL, & Zou L (2011). ATR: a master conductor of cellular responses to DNA replication stress. Trends in biochemical sciences, 36 (3), 133-40 PMID: Myers K, Gagou ME, Zuazua-Villar P, Rodriguez R, & Meuth M (2009). ATR and Chk1 suppress a caspase-3-dependent apoptotic response following DNA replication stress. PLoS genetics, 5 (1) PMID: 19119425 

Rawlinson SM, & Moseley GW (2015). The nucleolar interface of RNA viruses. Cellular microbiology, 17 (8), 1108-20 PMID: 26041433 

Wulan WN, Heydet D, Walker EJ, Gahan ME, & Ghildyal R (2015). Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses. Frontiers in microbiology, 6 PMID: 26082769 

Xu LH, Huang M, Fang SG, & Liu DX (2011). Coronavirus infection induces DNA replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of DNA polymerase δ. The Journal of biological chemistry, 286 (45), 39546-59 PMID: 21918226 

Wang, F., Fei, H., Cui, Y., Sun, Y., Li, Z., Wang, X., Yang, X., Zhang, J., & Sun, B. (2014). The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest, DNA damage response and autophagy in cancer cells Apoptosis, 19 (9), 1389-1398 DOI: 10.1007/s10495-014-1010-3 

Dove B, Brooks G, Bicknell K, Wurm T, & Hiscox JA (2006). Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication. Journal of virology, 80 (8), 4147-56 PMID: 16571830 

Liou JS, Wu YC, Yen WY, Tang YS, Kakadiya RB, Su TL, & Yih LH (2014). Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest. Toxicology and applied pharmacology, 278 (3), 249-58 PMID: 24823293 

Wrighton, K. (2015). Autophagy: Chaperone-mediated autophagy degrades CHK1 Nature Reviews Molecular Cell Biology, 16 (6), 328-328 DOI: 10.1038/nrm4003 

Filippi-Chiela EC, Villodre ES, Zamin LL, & Lenz G (2011). Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells. PloS one, 6 (6) PMID: 21695150 

Lee IH, Kawai Y, Fergusson MM, Rovira II, Bishop AJ, Motoyama N, Cao L, & Finkel T (2012). Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress. Science (New York, N.Y.), 336 (6078), 225-8 PMID: 22499945 

Wojciechowski J, Horky M, Gueorguieva M, & Wesierska-Gadek J (2003). Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells. International journal of cancer. Journal international du cancer, 106 (4), 486-95 PMID: 12845642 

Xu L, Khadijah S, Fang S, Wang L, Tay FP, & Liu DX (2010). The cellular RNA helicase DDX1 interacts with coronavirus nonstructural protein 14 and enhances viral replication. Journal of virology, 84 (17), 8571-83 PMID: 20573827 

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