Impairment of neurogenesis in ZIKV infected neuronal cells: strain specific ? Asian/American v. African strains

During the current Zika Virus (ZIKV) outbreaks in the Americas, an increased number of cognitive malformations including but not limited to microcephaly in foetuses and neonates of mothers who had been infected with ZIKV during pregnancy, lead to the conclusion that ZIKV might be neuroteratogenic,  a hypothesis that has been supported by findings that various ZIKV strains –including isolates from Asia such ZIKV SZ 01, ZIKV FSS13025 and H/PF/2013 as well as isolates from the Americas such as ZIKV Mex 1-144,  ZIKV PRV ABC059 and ZIKV BR Paraiba 2015 or the (original) African isolate ZIKV MR766- not only infect and replicate in neuronal cells in vitro and in vivo, but also induces apoptosis of infected and non-infected cells. These results suggest that ZIKV may cause abnormal neuronal development of the foetal brain by inducing cell death of infected cells via intrinsic apoptosis as well as bystander apoptosis of non-infected cells via the secretion of pro-inflammatory cytokines.

As described before, ZIKV MR766 infected human neuronal progenitor cells (hNPC), not only undergo apoptosis, but also are arrested at G2/M phase of the cell cycle as measured by single parameter flow cytometry, which is supported by findings that in ZIKV Mex 1-144 infected foetal brain tissue of mice a decrease in Ki67 positive and Histone H3-P (Ser-10) positive cells can be observed, suggesting that ZIKV infection arrests infected cells in G2 phase of the cell cycle. Further research however is need if ZIKV infection of proliferating neuronal cells also induces aberrant mitosis like Coronavirus infected Vero and primary chicken cells. Moreover, the infection of human i90c16 (induced pluripotent human neural stem cell that are derived from IMR-90 human lung fibroblast cells with ZIKV H/PF/2013 may induce genotoxic stress resulting in the formation of γH2AX (H2AX-Ser19) positive DNA damage repair foci; since in ZIKV MR766 infected hNPC genes encoding for proteins that are involved in the repair of DNA damage are downregulated, sustained presence of DNA damage might result in the activation of the G2 checkpoint, thus preventing mitotic entry. Change of the expression of genes related to the cellular DNA Damage Response (DDR), apoptosis and neurogenesis may be induced in a TLR-3 dependent manner since the infection of h9 derived human embryonic stem cells (hESC) with ZIKV MR766 induces the activation of TLR-3 and thus TLR-3 mediated pathways that downregulate the expression of genes related to neurogenesis and upregulation of genes related to apoptosis.

ZIKV BR AB_ES v. ZIKV MR766 induced apoptosis and the cell cycle 

In the original study published by Tang et al. in 2016, the authors infected hNPC derived from dermal fibroblasts with ZIKV MR766, the original ZIKV isolate from Uganda (1947) which was extensively passaged in mice in 1950s, and analysed the changes in the expression of genes at 72 hrs p.i. . As described in extensio before, ZIKV MR766 downregulates the expression of genes encoding proteins involved in the induction of DNA damage response pathways (HR, NHEJ and FA) as well as in the initiation of DNA replication such as MCM-6 and the progression of the cell cycle from G1 to S, S phase progression and mitosis. These results were confirmed in a study in which dermal derived hNPC were infected with ZIKV MR766 or ZIKV FSS13025 with gene expression analysed at 64 hrs p.i.. Interestingly, the latter study also identified genes involved in the repair of damaged DNA, DNA replication and cell cycle progression that are only downregulated in ZIKV FSS 13025 infected hNPC as well as genes that specifically upregulated in either ZIKV MR766 or ZIKV FSS 13025 infected hNPC, suggesting that different strains might alter the expression of a subset of genes in a strain specific manner. Indeed, only ZIKV strains of the Asian lineage, ZIKV FSS 13025 and ZIKV H/PF/2013 so far have been shown to induce p53.

The ZIKV strains that are currently circulating in the Americas are derived from the Asian ZIKV lineage with ZIKV circulating in Brazil being 97-100% similar to Asian isolates. The infection of hNPC derived from dermal fibroblasts with ZIKV BR AB_ES for 72 hrs results in widespread apoptosis concomitant with the activation of Caspase-3 and a reduction of the number of cells expressing the neuronal markers Sox-2 and HUC/D, indicating reduction in the growth of neurospheres, thus depleting the pool of neural progenitor cells. In contrast to ZIKV MR766 infected hNPC however, the infection of hNPC with ZIKV BR AB_ES does not induce a pronounced arrest in G2 phase of the cell cycle, suggesting that ZIKV BR AB_ES -in contrast to ZIKV H/PF/2013 infected hNPC- might induce apoptosis independent of cell cycle arrest. Further experiments are however needed to determine if this is truly the cases since it might be possible that infected cells arrest at G2 prior 72 hrs p.i.. Based on gene expression data, both ZIKV MR766 and ZIKV BR AB_ES downregulate the expression of Cyclin E2 suggesting that both in ZIKV MR766 and ZIKV BR AB_ES infected hNPC the assembly of the pre-replication complex at the DNA and the G1/S transition might be affected. In addition, infection of hNPC with either ZIKV isolate downregulates the expression of MCM-6, suggesting that DNA replication might be inhibited. In contrast to ZIKV MR766, ZIKV BR AB_ES infection of hNPC upregulates the expression of components of the DDR, namely FANCD2, BRCA1, and MRE11A as well inducing the expression of DRAM-1 (probably via the induction of p53), suggesting that at least some DDR pathways might be not affected by ZIKV BR AB_ES.

Figure: Differences between ZIKV BR AB_ES and ZIKV MR766: DNA replication and cell cycle 

In addition to differences in the expression pattern of genes related to the control of the cell cycle and the DDR, genes that have proposed to be involved in viral replication are upregulated in hNPC infected with ZIKV BR AB_ES compared with previously published data obtained from hNPC ZIKV MR766 infected cells.

Figure:  Differences between ZIKV BR AB_ES and ZIKV MR766: genes proposed to be involved in viral replication 

In conclusion, the infection of hNPC with ZIKV BR AB_ES or ZIKV MR766 induces apoptosis of hNPC at 72 hrs p.i. probably because of inducing either a G1/S arrest and/or G2 arrest of the cell cycle although mitotic abnormalities at least in a subset of infected cells cannot be ruled out.   These changes in cell cycle progression are accompanied by a downregulation of genes involved in the onset and progression of S phase and DNA replication, suggesting that both isolates from the African and the Asian ZIKV lineage induce either stalled replication forks or prevent the formation of DNA replication complexes. Indeed, CldU pulse labelling experiments of ZIKV Mex 1-144 infected foetal (mice) NPC display an extension of S phase and in ZIKV BR AB_ES infected hNPC the expression of CDKN1A is upregulated suggesting that the CyclinE-cdk2 complex is inhibited although this has not been tested using a H1-kinase assay.

The downregulation of the expression of several genes involved in the progress of mitosis in ZIKV MR766 infected hNPC suggests that the infection of hNPC with ZIKV might also prevent or prolong mitotic exit, promoting aberrant cytokinesis. ZIKV FB-GWUH-2016 and ZIKV H/PF/2013 infected hNPC exhibit mother centrioles that lack appendages as well as one to the triplet microtubular blades at the distal ends, potentially resulting in multiple centrosomes during mitosis and consequently in aberrant mitosis. Indeed the hNPC infected with ZIKV BR/Bahia exhibit abnormal chromosome number (aneuploidy 12 + 17, mono-somy 12/17 and trisomy 12/17) as well as  multi- and bipolar cell division followed by    formation of micronuclei. 

Figure Abnormal centrosomes in ZIKV AS/AM infected hNPC

In addition, the downregulation of Centriolin in ZIKV BR AB_ES and ZIKV MR766 infected hNPC suggests that infected hNPC might either undergo apoptosis due to arrested cytokinesis or be arrested in G1 phase of the cell cycle. This supported by findings that both ZIKV FB-GWUH-2016 and ZIKV H/PF/2013 infected) but not ZIKV MR766 infected hNPC exhibit impaired neurogenesis due to perturbed centrosomes. 




Similar to centrinone treated hTERT-RPE1 immortalized retinal pigment epithelial cells (RPE1 cells), the infection of hNPC with either ZIKV H/PF/2013, ZIKV FB-GWUH-2016 or ZIKV BR AB_ES might trigger G1 arrest via the induction of 53BP1 in a p53 dependent manner; interestingly, BRCA-1 has also been implicated in mediating G1/S arrest as well as Bax dependent apoptosis, suggesting that ZIKV might induce cell cycle arrest and apoptosis via multiple pathways.

   

Figure: Asian and American ZIKV isolates induce cell cycle arrest by different pathways 

Different infectious profiles of ZIKV strains were previously described for African (ZIKV ArB41644) and Asian (ZIKV H/PF/2013) infected human iPSc derived neural stem cells (NSC), dermal fibroblast derived hNPC infected with either ZIKV MR766 or ZIKV FSS 13025. So far however, no studies are available that analyse individual viral genes nor extensive studies that examine the progression of the cell cycle in synchronised cells infected with different ZIKV strains. Also, it remains to be seen if primary isolates of African strains are similar to ZIKV MR766 or resemble Asian isolates. Finally, while experiments in hNPC are important, similar experiments are needed in mosquitoe cells.


Further reading 

Li H, Saucedo-Cuevas L, Shresta S, & Gleeson JG (2016). The Neurobiology of Zika Virus. Neuron, 92 (5), 949-958 PMID: 27930910 

van den Pol AN, Mao G, Yang Y, Ornaghi S, & Davis JN (2017). Zika virus targeting in the developing brain. The Journal of neuroscience : the official journal of the Society for Neuroscience PMID: 28123079 

Li H, Saucedo-Cuevas L, Regla-Nava JA, Chai G, Sheets N, Tang W, Terskikh AV, Shresta S, & Gleeson JG (2016). Zika Virus Infects Neural Progenitors in the Adult Mouse Brain and Alters Proliferation. Cell stem cell, 19 (5), 593-598 PMID: 27545505 

Tang H, Hammack C, Ogden SC, Wen Z, Qian X, Li Y, Yao B, Shin J, Zhang F, Lee EM, Christian KM, Didier RA, Jin P, Song H, & Ming GL (2016). Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell stem cell, 18 (5), 587-90 PMID: 26952870 

Garcez PP, Nascimento JM, de Vasconcelos JM, Madeiro da Costa R, Delvecchio R, Trindade P, Loiola EC, Higa LM, Cassoli JS, Vitória G, Sequeira PC, Sochacki J, Aguiar RS, Fuzii HT, de Filippis AM, da Silva Gonçalves Vianez Júnior JL, Tanuri A, Martins-de-Souza D, & Rehen SK (2017). Zika virus disrupts molecular fingerprinting of human neurospheres. Scientific reports, 7 PMID: 28112162 

Gabriel, E., Ramani, A., Karow, U., Gottardo, M., Natarajan, K., Gooi, L., Goranci-Buzhala, G., Krut, O., Peters, F., Nikolic, M., Kuivanen, S., Korhonen, E., Smura, T., Vapalahti, O., Papantonis, A., Schmidt-Chanasit, J., Riparbelli, M., Callaini, G., Krönke, M., Utermöhlen, O., & Gopalakrishnan, J. (2017). Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids Cell Stem Cell DOI: 10.1016/j.stem.2016.12.005 

Souza BS, Sampaio GL, Pereira CS, Campos GS, Sardi SI, Freitas LA, Figueira CP, Paredes BD, Nonaka CK, Azevedo CM, Rocha VP, Bandeira AC, Mendez-Otero R, Dos Santos RR, & Soares MB (2016). Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells. Scientific reports, 6 PMID: 28008958

Ghouzzi VE, Bianchi FT, Molineris I, Mounce BC, Berto GE, Rak M, Lebon S, Aubry L, Tocco C, Gai M, Chiotto AM, Sgrò F, Pallavicini G, Simon-Loriere E, Passemard S, Vignuzzi M, Gressens P, & Di Cunto F (2017). ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53. Cell death & disease, 8 (1) PMID: 28102842 

Caldon CE, & Musgrove EA (2010). Distinct and redundant functions of cyclin E1 and cyclin E2 in development and cancer. Cell division, 5 PMID: 20180967 

Gromley A, Jurczyk A, Sillibourne J, Halilovic E, Mogensen M, Groisman I, Blomberg M, & Doxsey S (2003). A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase. The Journal of cell biology, 161 (3), 535-45 PMID: 12732615

Shimada M, Matsuzaki F, Kato A, Kobayashi J, Matsumoto T, & Komatsu K (2016). Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly. PloS one, 11 (7) PMID: 27367050 

Meitinger F, Anzola JV, Kaulich M, Richardson A, Stender JD, Benner C, Glass CK, Dowdy SF, Desai A, Shiau AK, & Oegema K (2016). 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. The Journal of cell biology, 214 (2), 155-66 PMID: 27432897 

Liu H, Gong M, French BA, Liao G, Li J, Tillman B, & French SW (2015). Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing. Oncotarget, 6 (40), 42491-503 PMID: 26623723