ZIKV: ATM dependent signalling, VRK1, autophagy and the ER stress response in neuronal cells

Upon the induction of DNA damage, complex signaling pathways are activated that regulate the ability of cells to detect and repair the damage since both single and double strand DNA damage pose significant risk to cell survival and transmission of unrepaired DNA damage to progeny is associated not only with aging and cancer but also with neurodegenerative diseases. During the DNA damage response (DDR) ds and ss DNA breaks are recognised by ATM, ATR and DNA-PK kinases, which in turn activate signaling pathways that converge on p53 and other scaffold proteins such as 53BP1, that upon recruitment are localised at DNA repair foci. Nuclear Vaccinia related kinase-1 (VRK1) is a nuclear Ser/Thr kinase that phosphorylates multiple proteins involved in the DDR –including p53 and 53BP1- as well as promoting the entry of cells into mitosis by phosphorylating Histone H3 at Thr-3 and Ser-10, thus promoting nuclear condensation.

Figure: Functions of VRK1 in mitotic entry

Figure: Functions of VRK1 in ATM mediated signalling during DDR

In the case of p53, VRK1 stabilises p53 by phosphorylating p53 at Thr-18 thus increasing p53 dependent gene expression and preventing the degradation of p53 by MDM2. During the DDR, VRK1 is predominantly associated with chromatin remodeling and recruitment of 53BP1 to sites of DNA damage and promoting phosphorylation of H2AX at Ser139 in a ATM and p53 dependent pathway as well as promoting the acetylation of both Histone H3 and H4 by recruitment of p300/CBP.

In human cells, loss of VRK1 is associated with arrest in G0 but not G2 phase of the cell cycle and mutations of VRK1 have been associated with complex motor and sensory axonal neuropathy and microcephaly. Since in both ZIKV infected mice brain cells and ZIKV infected human neuronal progenitor cells (hNPC) VKR1 expression is decreased, this supports the notion that the observed defects in the neuronal defects are due to mitotic defects induced by ZIKV.

Figure and table: Gene changes in ZIKV infected foetal cells regarding components of
the non canonical ULK signalling and VRK

Figure: ZIKV and VRK1:inhibition of Histone H# phosphorylation 

In addition to promote the DDR and mitotic entry, the activation of VRK1 by Polo-like kinase 3 (Plk3) is also required for MEK1 dependent fragmentation of the Golgi during mitosis and the entry of cells into S phase by inducing the expression of Cyclin D in CREB dependent manner; ZIKV mediated downregulation of VRK1 therefore might not only prevent mitotic entry and the DDR but also interferes with the fragmentation of the Golgi as well as entry of infected cells into S phase. As described before, inhibition of mitotic entry by ZIKV has been proposed to be associated with a prolonged S phase as evidenced by an increase of BrdU positive cells in ZIKV infected foetal brain cells. If this is the case, then ZIKV infection of G1 cells might either not downregulate cyclin D1 expression per se but the observed decrease of Cyclin D expression might be associated with an increase of S phase cells instead. Decreased levels of VKR1 therefore might therefore primarily associated with preventing mitotic entry. Further studies are therefore needed to determine the pathways associated with prolonging S phase v. preventing mitotic entry of ZIKV infected cells.   

Figure: ZIKV, VRK1 and Cyclin D1

Additionally, VRK1 also induces the degradation of p53 in a DRAM1 dependent pathway via autophagy; therefore, ZIKV might promote the degradation of p53 via Mdm2 dependent ubiquitination and subsequent proteasomal degradation of p53.

Figure: ZIKV, ATM, VRK1, and p53: ZIKV may increase degradation of p53 via the proteasome
and inhibit DRAM1 dependent autophagy

Phosphorylation of ATM at Ser-181 and ATM dependent formation of autophagosomes however can not only be induced by DDR signaling but also ER stress signaling pathways, namely by CHOP. Indeed, the infection of MDCK and HeLa GFP-LC3 cells with Dengue Virus 2 (DENV2) induces the formation of autophagosomes in the absence of apoptosis in a ATM dependent manner since the inhibition of ATM using Caffeine or siATM both increased the sensitivity of DENV2 infected MDCK cells to Camptothecin as well as decreasing levels of LC3-II (autophagic flux was not determined) at 24 hrs p.i. . During later stages of infection, ATM is is induced in a ROS dependent manner which either are accumulating due to increased PERK activity or mitochondrial damage.

In the case of ZIKV, ATM therefore could be induced as a result of stalled replication as proposed before, the induction of the ER stress response or (especially at late stages of the replication cycle) due to the production of mitochondrial ROS as a result of mitochondrial damage.

Despite the increase in the formation of autophagosomes, autophagic flux in both DENV 2 and ZIKV infected cells however might be inhibited; in the case of DENV, p62/SQSTM1 is degraded by the proteasome and in ZIKV infected hNPC the expression of LAMP2 is downregulated.

In addition, in ZIKV infected cells the clearance of misfolded proteins that accumulate in the ER might be prevented by inhibition of ER-to Golgi COPII dependent traffic and thus contribute to neuronal death similar to ULK1/2 double knockout mice. In ULK1/2 double knockout mice, a complex consisting of SEC16A, SEC23 and SEC24A is activated by site specific of SEC16A by the cellular ULK1/2, thus localising the complex to ER exit sites and promoting the clearance of cargo via COPII dependent traffic. In order to investigate if ZIKV disrupts this specific pathway however, neuronal cells need to be used since in other cell lines this noncanonical role of ULK1/2 might not play an essential role in the clearance of accumulated protein.

In conclusion, ZIKV might activate ATM upon DNA damage and/or the induction of the ER stress response. In both cases, this response however might be abrogated due to the downregulation of VKR1 and components of the COPII dependent ER to Golgi trafficking pathway, leading to neuronal death.

Further reading

Lamarche, B., Orazio, N., & Weitzman, M. (2010). The MRN complex in double-strand break repair and telomere maintenance FEBS Letters, 584 (17), 3682-3695 DOI: 10.1016/j.febslet.2010.07.029

Kang TH, Park DY, Kim W, & Kim KT (2008). VRK1 phosphorylates CREB and mediates CCND1 expression. Journal of cell science, 121 (Pt 18), 3035-41 PMID: 18713830  

Lopez-Sanchez, I., Sanz-Garcia, M., & Lazo, P. (2008). Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser342, a Downstream Target in a Pathway That Induces Golgi Fragmentation Molecular and Cellular Biology, 29 (5), 1189-1201 DOI: 10.1128/MCB.01341-08 

Gonzaga-Jauregui C, Lotze T, Jamal L, Penney S, Campbell IM, Pehlivan D, Hunter JV, Woodbury SL, Raymond G, Adesina AM, Jhangiani SN, Reid JG, Muzny DM, Boerwinkle E, Lupski JR, Gibbs RA, & Wiszniewski W (2013). Mutations in VRK1 associated with complex motor and sensory axonal neuropathy plus microcephaly. JAMA neurology, 70 (12), 1491-8 PMID: 24126608 

Kang TH, Park DY, Choi YH, Kim KJ, Yoon HS, & Kim KT (2007). Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells. Molecular and cellular biology, 27 (24), 8533-46 PMID: 17938195 

 Salzano M, Sanz-García M, Monsalve DM, Moura DS, & Lazo PA (2015). VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage. Epigenetics, 10 (5), 373-83 PMID: 25923214 

Datan E, Roy SG, Germain G, Zali N, McLean JE, Golshan G, Harbajan S, Lockshin RA, & Zakeri Z (2016). Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation. Cell death & disease, 7 PMID: 26938301 

Metz P, Chiramel A, Chatel-Chaix L, Alvisi G, Bankhead P, Mora-Rodriguez R, Long G, Hamacher-Brady A, Brady NR, & Bartenschlager R (2015). Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62. Journal of virology, 89 (15), 8026-41 PMID: 26018155 

Joo, J., Wang, B., Frankel, E., Ge, L., Xu, L., Iyengar, R., Li-Harms, X., Wright, C., Shaw, T., Lindsten, T., Green, D., Peng, J., Hendershot, L., Kilic, F., Sze, J., Audhya, A., & Kundu, M. (2016). The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis Molecular Cell, 62 (4), 491-506 DOI: 10.1016/j.molcel.2016.04.020