Virology tidbits

Virology tidbits

Wednesday 14 September 2016

ZIKV: antivirals and the cell cycle, TBK-1 relocalisation and immune signalling



Zika Virus (ZIKV) is an emerging flavivirus that was first isolated in 1947 from a sentinel monkey in Uganda as part of study that aimed to identify novel pathogens and despite sporadic local outbreaks in countries such as Gabon, Nigeria, Cambodia, Malaysia and Indonesia followed by the first major outbreak in Yap/Federal States of Micronesia 2007  only caused mild disease in humans with up to 80% of asymptomatic cases.

The emergence of ZIKV combined with severe pathogenicity following the outbreak in French Polynesia 2013/2014 with an excess of 30000 patients and particular the introduction of ZIKV to Brazil  as early as 2013 as suggested by molecular clock analysis however raised questions about the molecular evolution of ZIKV since ZIKV was previously only associated with arthralgia and a mild febrile illness but not neuropathological disorders including abnormal foetal brain development and Guillain-Barre Syndrome (GBS) that were first identified in Pernambuco/Brazil and in a retroactive study of the 2013 outbreak in French Polynesia.

ZIKV is a flavivirus closely related to Dengue Virus (DENV), Japanese Encephalitis Virus (JEV) and Yellow Fever Virus (YFV) with a single stranded positive stranded RNA genome of approximately 10800 bp. Similar to DENV, JEV and YFV, the ZIKV RNA encodes for a single polyprotein that it is cleaved into the structural (Capsid (C), pre-membrane (prM), and envelope (E)) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5) proteins with the replication taking place in the cytoplasm of infected although at least the C and NS5 proteins localise to the nucleolus and to nuclear speckles respectively, suggesting that the nuclear localisation of these proteins might be required for efficient replication of JEV probably due to the interaction of the JEV core protein with B23, thus relocalising B23 to the nuclear periphery. In contrast to JEV core protein however, the DENV core protein does not co-localize with B23. In the case of NS5, the expression of DENV NS5 interacts with components of the cellular spliceosome –in particular with components of the U5 small nuclear ribonucleoprotein particle- and thus disrupts the maturation of cellular pre-mRNA by decreasing the efficiency of pre-mRNA processing, thus contributing to the downregulation of cellular gene expression. Presently it is not known to which extent ZIKV derived proteins interfere with these processes.

Both DENV and ZIKV NS5 have been shown to inhibit the nuclear translocation of STAT2 and thus antiviral signaling, suggesting that ZIKV and DENV NS5 exhibit similar if not identical properties and similar to DENV, so called “viral factories” or viral replication centers are formed in the cytoplasm of ZIKV infected cells which contain both viral (progeny) RNA as well as viral proteins. Since ZIKV RC are similar to the viral replication centers of other positive strand RNA viruses and are positive for LC3, it has been proposed that these are formed by utilizing the autophagic machinery although in A549 cells infected with the South Pacific ZIKV PF-25013-18 no LC3-B positive structures have been identified (in contrast with ZIKV 766 infected human keratinocytes or ZIKV SPH 2015 infected human astrocytes) and chloroquine inhibits ZIKV replication in infected U87 glioblstoma cells.

In any case, as mentioned above, both the ZIKV outbreak in French Polynesia and the current outbreak in the Americas are are associated with neurological abnormalities, namely foetal microcephaly/micrencephaly, lissencephaly, hydrocephaly, cortical/periventricular calcifications, hypoplasia of the brain stem and spinal cord, necrosis  and other congenital abnormalities such as focal pigment mottling of the retina, optic nerve abnormalities and chorioretinal atrophy in foetuses and newborns of previously infected women as well as uveitis, conjunctivitis and GBS in adults. These observations suggest that ZIKV is neurotrophic, a finding which was first reported in mice following the isolation of ZIKV from the sentinel monkey (for further details see previous discussion here). Subsequent studies demonstrated that ZIKV enters neuronal and non-neuronal cells via different receptors, the phosphatidylserine TAM receptor Axl that is enriched on the surface of human glial cells and the main receptor, and with DC-SIGN, TIM-1 and Tyro-3 as minor receptors.
Consequently, recently published studies which have been discussed in extensio before, suggest that ZIKV can infect human neural progenitor cells (hNPC) derived from induced pluripotent stem cells, brain organoids and neurospheres that are derived from embryonic stem cells or induced pluripotent stem cells as well as two foetal cell lines. These studies showed that ZIKV induces caspase-3 dependent apoptosis which may be preceded by mitochondrial depolarization and subsequent activation of caspase-3 via the release of cytochrome-c although the mechanism leading to mitochondrial depolarisation has not been elucidated (see previous post for discussion). ZIKV infected foetal neural tissue samples derived 13-16 weeks post conception exhibits high levels of infection in the ventricular and subventricular zone which are positive for radial glial cells  with only a small number of mature neurons being infected and later (18 weeks pcw), suggesting that postmitotic neurons are not susceptible to ZIKV which is confirmed by the absence of Axl in mature neurons.

More recent studies also identified the vaginal mucosa and lacrimal glands of mice as being susceptible for ZIKV thus providing a model of sexual transmission and viral persistence respectively. Interestingly, ZIKV infection of the adult neurosensory retina induces apoptosis as measured by TUNEL staining yet does not induce significant pan-retinal abnormalities.

Antiviral drugs: targeting caspase-3

Recently, a drug repurposing screen identified several small molecule inhibitors that inhibit ZIKV induced caspase-3 dependent apoptosis in ZIKV FSS 13025 (Cambodia 2010) or ZIKV MR766 (Uganda 1947) infected SNB-19 glioblastoma, human astrocytes and hNPC. In this assay, 194 compounds were tested using two high-throughput assays with one measuring both cell viability at 72 hrs p.i. and caspase-3/-7 activity at 6 hrs p.i. and the other measuring the caspase-3/-7 activity in a primary screen followed secondary screen measuring both cell viability and caspase-3/-7 activity which is then followed by tertiary screen that involves a ZIKV replication assay, 2D & 3D neural cell models (such as hNPC and brain organoids) and in addition measuring the effect of drug combinations on ZIKV replication and cell viability. Despite causing apoptosis in all cell types tested, ZIKV MR766 induced apoptosis can only be prevented by 35 compounds tested in all cell types, with 54 inhibiting apoptosis in human astrocytes, 57 in SNB-19 glioblastoma cells and 48 in hNPC, whereas only 1 compound –a pan-caspase inhibitor (Emericasan)- inhibiting both caspase-3/-7 activity and increasing the viability of ZIKV MR766, ZIKV FSS 13025 and ZIKV PRVABC59 infected hNPC/SNB-19 cells and brain organoids. In contrast to Emericasan, the vast majority of screened not only inhibited apoptosis but also had a negative impact on cell proliferation even in the absence of viral infection.

Figure: Effect of tested compounds on cell viability of ZIKV infected cells (Astrocytes, SNB-19 glioblastoma cells
and hNPC) Negative cell viablity=toxic effect even in absence of ZIKV 

Figure: Effect of tested compounds on ZIKV induced caspase-3 activity 


Besides preventing ZIKV induced apoptosis, Emericasan also reduced viral replication as measured by determining viral titres and measuring the expression levels of the viral NS1 protein, suggesting that the inhibition of cellular caspases also inhibits viral replication. One possibility is that ZIKV induced activation of caspase-3/-7 and/or other caspases inactivates Beclin-1 induced autophagy by cleaving Beclin-1 at AA 133 and AA149 (TDVD133 and DQLD149 respectively) thus not only preventing autophagy but also localising the resulting Beclin-1 C terminal fragment to the mitochondria, inducing the release of Cytochrome-c in addition to cleaving Phosphatidylinositol-3-Kinase (PI3KC3)/vacuolar protein sorting complex-34 (Vps-34). Restoring Beclin-1 dependent autophagy in Emericasan treated and ZIKV infected cells therefore might contribute to the inhibition of viral replication.



Additionally, the replication of ZIKV FSS13025 and ZIKV PRVABC59 in SNB-19 cells and human astrocytes can be efficiently inhibited by Cyclin dependent kinase inhibitors (Cdki) such as PHA-690509, Niclosamide  and Seliciclib that inhibit the progression of the cell cycle, indicating that cellular Cdk might phosphorylate the viral NS5 and/or NS5a protein similar to the DENV-2, TBE  and YFV NS5 or BVDV NS5a or that the progression in particular from the G1 phase of the cell cycle to S phase might be required for efficient ZIKV replication similar to Mouse Hepatitis Virus (MHV), Infectious Bronchitis Virus (IBV), SARS-CoV and Coxsackievirus B1. Further experiments are however needed to determine the role of Cdk’s in ZIKV replication which might involve using Cdk -/- MEF and/or siRNA targeting specific Cdk. The disadvantage of using Cdki however is that the proliferation of ZIKV PRVABC59 infected/Cdki treated cells as measured by EdU incorporation is significantly decreased compared to non-infected hNPC at 72 hrs p.i. thus limiting the use in utero. Cdki however might be useful in treating adults, preventing sexual transmission and/or prolonged shedding of ZIKV in urine, saliva and tears.



Figure: (A) Niclosamide: targeting viral entry (B) Cdki: targeting multiple cdk


ZIKV and the cell cycle: G2 and mitotic arrest

As discussed in a previous post, the in utero infection of (mouse) foetal brains with ZIKV SZ01 decreases the expression of proteins that previously have been linked to the development of microcephaly, in particular those that are involved in the separation of chromosomes during metaphase and anaphase, suggesting that ZIKV infected embryonic and/or foetal cells might exhibit incomplete cytokinesis and subsequent apoptosis, a notion that is supported by previous observations that  hNPC infected with ZIKV MR766 also exhibit a decrease in the expression of the very same genes confirming that the downregulation of genes regulating mitotic progression might arrest infected cells in G2/M phase of the cell cycle which is confirmed by flow cytometry analysis of infected hNPC. In addition to hNPC, more recent data indicate that the infection of neuroepithelial cells derived from the Neocortex (NCX-NES) derived from human specimens ranging from 5 to 8 weeks postconception with ZIKV FSS 13025 not only support viral replication as evidenced by the expression of the viral NS1 protein but also undergo caspase-3 dependent apoptosis including nuclear fragmentation and pyknosis as well exhibiting decreased cell proliferation as indicated by the absence of the proliferation marker Ki-67 starting at day 3.5 p.i. and continuing until day 6.5 p.i. . In contrast to NCX-NES cells, mature neurons do not support viral replication as measured by the presence of NS1 probably due to the absence of the entry receptor, Axl, and do not show a significant increase in apoptosis. Similar to the ZIKV SZ01 isolate, ZIKV FSS13025 and the ZIKV BR 243 strain (derived from the current outbreak in Brazil) also infect radial glial cells (RGC) of the ventricular zone (VZ), subventricular zone (SVZ) and the intermediate zone (IZ/SP), all of which contain PCNA positive proliferating cells and VIM positive RGC cells, of ex vivo foetal brain slices with viral replication being detected as early as day 3.5 p.i. , similar to NCX-NES cells. Most interestingly however, only ZIKV infected and ZIKV NS1 positive cells exhibit an aberrant cell morphology which indicates that primary proliferating neuronal cells infected with an ZIKV replication competent strain (but not with an UV inactivated strain) induce a cell cycle arrest.
In addition to downregulating the expression of genes related to mitotic progression such as Aurora Kinase-B, activation of the innate immune response can induce apoptosis, i.e. via IRF-3 mediated activation of Bax by Sendai Virus (SeV). In this case, the dsRNA intermediate activates IPS-1 which in turn recruits TANK-binding Kinase-1 (TBK-1) which in turn phosphorylates and activates IRF-3, the latter binding Bax and translocating to the mitochondrion.
In the case of ZIKV infected NCX-NES cells or foetal brain slices neither ZIKV FSS13025 nor ZIKV PE243 increases the expression of TBK-1 but rather relocalises TBK-1 from the centrosome to mitochondria thus potentially preventing the phosphorylation of the centrosomal protein CEP170 and the mitotic apparatus protein NuMA as well as g-tubulin, leading to mitotic defects such as aberrant cytokinesis characterised by the presence of a cleavage furrow in G1 phase of the cell cycle and/or subsequent apoptosis due to mitotic catastrophe. The presence of cells with a cleavage furrow might also explain the presence of a small percentage of ZIKV positive (postmitotic) neurons.

In addition, inhibiting the progress of mitosis, TBK-1 also might recruit IRF-3 and Bax to the mitochondria thus providing an alternative pathway culminating in apoptosis independent of mitotic arrest. Further experiments are needed to distinguish both pathways. Paradoxically the inhibition of TBK-1 with inhibitors such as BX795 or Amlexanox exacerbates ZIKV induced apoptosis. One reason might be that the relocalisation of TBK-1 in ZIKV infected cells also induces mitophagy by recruiting p62/SQSTM-1 and/or optineurin so that treating infected cells with TBK-1 also decreases mitophagy and thus the clearance of damaged mitochondria.
Besides the induction of apoptosis, mitochondrial localisation of TBK-1 may also interfere with immune signalling by disrupting STING mediated phosphorylation of TBK-1 at perinuclear granulae and thus the translocation of IRF-3 to the nucleus, thus subsequently inhibiting the Interferon response. In this context, results from both WNV and DENV-1, -2 and -4 infected primary endothelial cells and HEK 293T cells indicate that the viral encoded NS2A and NS4B inhibit the phosphorylation of both TBK-1 and IRF-3 and subsequent induction of Interferon-beta, with DENV-1/-2/-4 NS4A uniquely inhibiting TBK-1 and IKKε-directed signalling. In a similar way, PEDV has been shown to inhibit TBK-1 signalling as well. Additionally, the expression of ZIKV NS4B protein might –similar to to DENV NS4B- induce the elongation of mitochondria in infected cells and disrupting the ER-Mitochondiria contact (MAM) which is critical for immune signalling and thus abrogating the celluar RIG-1 dependent interferon response.

Figure: ZIKV and DENV-1/-2/-4 NS4A and TBK-1 mediated signalling:
targeting phosphorylation of IRF-3


Apart from inhibiting mitotic progression, interfering with TBK-1 dependent immune signalling  and inducing apoptosis, mitochondrial TBK-1 might also induce lipophagy and thus promote viral replication by inducing mitophagy via recruitment of p62/SQSTM-1, NDP52 and/or Optineurin. Again further studies are warranted.

In conclusion, mitotic arrest of ZIKV infected primary neuronal cells might be caused by multiple reasons. As discussed before, based on gene expression analysis of ZIKV MR766 infected hNPC ZIKV might induce DNA replication stress that ultimately induces cell cycle arrest and aberrant mitosis; if this cell cycle delay is preceded by a prolonged S phase similar to IBV has not been determined. Mitotic progression of ZIKV infected hNPC might also be delayed by downregulating components of the mitotic machinery such as Aurora-A/B, components required for chromosome segregation or the Anaphase Promoting Complex/Cyclosome (APC/C). ZIKV induced activation of autophagy might also inhibit the onset of mitosis and thus arrest cells in G2 phase of the cell cycle in addition to inhibiting the progression of S to G2 and/or G2 progression. Again, further studies using EdU or BrdU incorporation to determine cell cycle progression in ZIKV infected cells are needed (both synchronised and non-synchronised cells).


Table: Genes related to mitotic progression that are up- or downregulated in ZIKV MR766 infected hNPC


The inhibition of viral replication by Cdki indicates that Cdk’s are essential for viral replication and further studies using siRNA and/or specific inhibitors should clarify the contribution of individual Cdk such as Cdk-4/-6, Cdk-2, Cdk-3, Cdk-1 and -since ZIKV infects neuronal cells- also Cdk-5. The latter is of particular interest since the inhibition of Cdk-5 has been shown to confer protection against neuronal apoptosis of cerebral granule neurons and prevent aberrant S-phase entry of postmitotic neurons.  
In addition to (potentially) phosphorylating viral proteins, Cdk might facilitate ZIKV replication indirectly by creating favourable conditions for viral entry. In the case of DENV-2 and DENV-3, HepG2 cells have been demonstrated to be more permissive for both infection and viral replication in G2 phase of the cell cycle compared to G1 or S phase which might explain why the combination of Niclosamide and a Cdki increases the cell viability of ZIKV infected human astrocytes and hNPC as well as decreasing viral replication.


Inhibiting caspase-3 dependent apoptosis also might prevent the cleavage of Beclin-1 and thus promote autophagy and increase cell viability in addition in preventing mitotic entry. It is crucial therefore to determine if caspase-3 inhibition has a negative effect on cell proliferation. Based on the results obtained from treating ZIKV infected astrocytes with Cdki, it may be possible that despite limiting ZIKV replication and increasing cell viability pan-caspase inhibitors might not allow the proliferation of primary neuronal cells infected with ZIKV.
Finally, similar to Porcine Respiratory Syndrome Virus (PRRSV) induced apoptosis, following the initial activation of caspase-3, the cleavage of Beclin-1 in ZIKV infected cells might enhance mitochondrial depolarisation by releasing the pro-apoptotic BH3 only protein Bad, followed by the localisation of Bad to the mitochondria where it forms a dimer with the anti-apoptotic Bcl-XL thus inactivating Bcl-XL.  Since so far cleavage of Beclin-1 in ZIKV infected cells has not been demonstrated, it remains to be seen if this is the case or not.


Figure: Hypothetical network of ZIKV induced changes to the cell cycle in infected cells



ResearchBlogging.org

Further reading

Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G, Soto-Acosta R, Galarza-Muñoz G, McGrath EL, Urrabaz-Garza R, Gao J, Wu P, Menon R, Saade G, Fernandez-Salas I, Rossi SL, Vasilakis N, Routh A, Bradrick SS, & Garcia-Blanco MA (2016). A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell host & microbe, 20 (2), 259-70 PMID: 27476412

Bonaldo MC, Ribeiro IP, Lima NS, Dos Santos AA, Menezes LS, da Cruz SO, de Mello IS, Furtado ND, de Moura EE, Damasceno L, da Silva KA, de Castro MG, Gerber AL, de Almeida LG, Lourenço-de-Oliveira R, Vasconcelos AT, & Brasil P (2016). Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil. PLoS neglected tropical diseases, 10 (6) PMID: 27341420

Campos Rde M, Cirne-Santos C, Meira GL, Santos LL, de Meneses MD, Friedrich J, Jansen S, Ribeiro MS, da Cruz IC, Schmidt-Chanasit J, & Ferreira DF (2016). Prolonged detection of Zika virus RNA in urine samples during the ongoing Zika virus epidemic in Brazil. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 77, 69-70 PMID: 26921737


Chatel-Chaix L, Cortese M, Romero-Brey I, Bender S, Neufeldt CJ, Fischl W, Scaturro P, Schieber N, Schwab Y, Fischer B, Ruggieri A, & Bartenschlager R (2016). Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses. Cell host & microbe, 20 (3), 342-56 PMID: 27545046

Costa F, Sarno M, Khouri R, de Paula Freitas B, Siqueira I, Ribeiro GS, Ribeiro HC, Campos GS, Alcântara LC, Reis MG, Weaver SC, Vasilakis N, Ko AI, & Almeida AR (2016). Emergence of Congenital Zika Syndrome: Viewpoint From the Front Lines. Annals of internal medicine, 164 (10), 689-91 PMID: 26914810

Cugola FR, Fernandes IR, Russo FB, Freitas BC, Dias JL, Guimarães KP, Benazzato C, Almeida N, Pignatari GC, Romero S, Polonio CM, Cunha I, Freitas CL, Brandão WN, Rossato C, Andrade DG, Faria Dde P, Garcez AT, Buchpigel CA, Braconi CT, Mendes E, Sall AA, Zanotto PM, Peron JP, Muotri AR, & Beltrão-Braga PC (2016). The Brazilian Zika virus strain causes birth defects in experimental models. Nature, 534 (7606), 267-71 PMID: 27279226

DICK GW (1953). Epidemiological notes on some viruses isolated in Uganda; Yellow fever, Rift Valley fever, Bwamba fever, West Nile, Mengo, Semliki forest, Bunyamwera, Ntaya, Uganda S and Zika viruses. Transactions of the Royal Society of Tropical Medicine and Hygiene, 47 (1), 13-48 PMID: 13077697


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


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

Garcez PP, Loiola EC, Madeiro da Costa R, Higa LM, Trindade P, Delvecchio R, Nascimento JM, Brindeiro R, Tanuri A, & Rehen SK (2016). Zika virus impairs growth in human neurospheres and brain organoids. Science (New York, N.Y.), 352 (6287), 816-8 PMID: 27064148

Hanners NW, Eitson JL, Usui N, Richardson RB, Wexler EM, Konopka G, & Schoggins JW (2016). Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects. Cell reports, 15 (11), 2315-22 PMID: 27268504


Jurado KA, Simoni MK, Tang Z, Uraki R, Hwang J, Householder S, Wu M, Lindenbach BD, Abrahams VM, Guller S, & Fikrig E (2016). Zika virus productively infects primary human placenta-specific macrophages. JCI insight, 1 (13) PMID: 27595140

Lazear HM, Govero J, Smith AM, Platt DJ, Fernandez E, Miner JJ, & Diamond MS (2016). A Mouse Model of Zika Virus Pathogenesis. Cell host & microbe, 19 (5), 720-30 PMID: 27066744

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 PMID: 27545505

Miner JJ, & Diamond MS (2016). Understanding How Zika Virus Enters and Infects Neural Target Cells. Cell stem cell, 18 (5), 559-60 PMID: 27152436

Miner JJ, Sene A, Richner JM, Smith AM, Santeford A, Ban N, Weger-Lucarelli J, Manzella F, Rückert C, Govero J, Noguchi KK, Ebel GD, Diamond MS, & Apte RS (2016). Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears. Cell reports, 16 (12), 3208-18 PMID: 27612415

Nowakowski TJ, Pollen AA, Di Lullo E, Sandoval-Espinosa C, Bershteyn M, & Kriegstein AR (2016). Expression Analysis Highlights AXL as a Candidate Zika Virus Entry Receptor in Neural Stem Cells. Cell stem cell, 18 (5), 591-6 PMID: 27038591

Oehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F, Baudouin L, Mallet H, Musso D, & Ghawche F (2014). Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December 2013. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 19 (9) PMID: 24626205

Onorati M, Li Z, Liu F, Sousa AM, Nakagawa N, Li M, Dell'Anno MT, Gulden FO, Pochareddy S, Tebbenkamp AT, Han W, Pletikos M, Gao T, Zhu Y, Bichsel C, Varela L, Szigeti-Buck K, Lisgo S, Zhang Y, Testen A, Gao XB, Mlakar J, Popovic M, Flamand M, Strittmatter SM, Kaczmarek LK, Anton ES, Horvath TL, Lindenbach BD, & Sestan N (2016). Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia. Cell reports, 16 (10), 2576-92 PMID: 27568284

Paploski IA, Prates AP, Cardoso CW, Kikuti M, Silva MM, Waller LA, Reis MG, Kitron U, & Ribeiro GS (2016). Time Lags between Exanthematous Illness Attributed to Zika Virus, Guillain-Barré Syndrome, and Microcephaly, Salvador, Brazil. Emerging infectious diseases, 22 (8), 1438-44 PMID: 27144515

Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, Hamersky GR, Jacob F, Zhong C, Yoon KJ, Jeang W, Lin L, Li Y, Thakor J, Berg DA, Zhang C, Kang E, Chickering M, Nauen D, Ho CY, Wen Z, Christian KM, Shi PY, Maher BJ, Wu H, Jin P, Tang H, Song H, & Ming GL (2016). Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell, 165 (5), 1238-54 PMID: 27118425 

Ribeiro LS, Marques RE, Jesus AM, Almeida RP, & Teixeira MM (2016). Zika crisis in Brazil: challenges in research and development. Current opinion in virology, 18, 76-81 PMID: 27179929

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 

Weaver SC, Costa F, Garcia-Blanco MA, Ko AI, Ribeiro GS, Saade G, Shi PY, & Vasilakis N (2016). Zika virus: History, emergence, biology, and prospects for control. Antiviral research, 130, 69-80 PMID: 26996139 

Wu KY, Zuo GL, Li XF, Ye Q, Deng YQ, Huang XY, Cao WC, Qin CF, & Luo ZG (2016). Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice. Cell research, 26 (6), 645-54 PMID: 27174054 

Xu, M., Lee, E., Wen, Z., Cheng, Y., Huang, W., Qian, X., TCW, J., Kouznetsova, J., Ogden, S., Hammack, C., Jacob, F., Nguyen, H., Itkin, M., Hanna, C., Shinn, P., Allen, C., Michael, S., Simeonov, A., Huang, W., Christian, K., Goate, A., Brennand, K., Huang, R., Xia, M., Ming, G., Zheng, W., Song, H., & Tang, H. (2016). Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen Nature Medicine DOI: 10.1038/nm.4184 

Zhang F, Hammack C, Ogden SC, Cheng Y, Lee EM, Wen Z, Qian X, Nguyen HN, Li Y, Yao B, Xu M, Xu T, Chen L, Wang Z, Feng H, Huang WK, Yoon KJ, Shan C, Huang L, Qin Z, Christian KM, Shi PY, Xu M, Xia M, Zheng W, Wu H, Song H, Tang H, Ming GL, & Jin P (2016). Molecular signatures associated with ZIKV exposure in human cortical neural progenitors. Nucleic acids research PMID: 27580721 

Rohn TT, Wirawan E, Brown RJ, Harris JR, Masliah E, & Vandenabeele P (2011). Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer's disease brain. Neurobiology of disease, 43 (1), 68-78 PMID: 21081164 

Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, Roelandt R, De Rycke R, Verspurten J, Declercq W, Agostinis P, Vanden Berghe T, Lippens S, & Vandenabeele P (2010). Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell death & disease, 1 PMID: 21364619 

Zhou A, Li S, Khan FA, & Zhang S (2016). Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction. Virulence, 7 (2), 98-109 PMID: 26670824 

Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, & Chen Q (2010). Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein & cell, 1 (5), 468-77 PMID: 21203962 Dalrymple, N., Cimica, V., & Mackow, E. (2015). Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant mBio, 6 (3) DOI: 10.1128/mBio.00553-15

Phoolcharoen W, & Smith DR (2004). Internalization of the dengue virus is cell cycle modulated in HepG2, but not Vero cells. Journal of medical virology, 74 (3), 434-41 PMID: 15368519 

Thepparit C, & Smith DR (2004). Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor. Journal of virology, 78 (22), 12647-56 PMID: 15507651

Chattopadhyay S, Marques JT, Yamashita M, Peters KL, Smith K, Desai A, Williams BR, & Sen GC (2010). Viral apoptosis is induced by IRF-3-mediated activation of Bax. The EMBO journal, 29 (10), 1762-73 PMID: 20360684 

Sharif-Askari E, Nakhaei P, Oliere S, Tumilasci V, Hernandez E, Wilkinson P, Lin R, Bell J, & Hiscott J (2007). Bax-dependent mitochondrial membrane permeabilization enhances IRF3-mediated innate immune response during VSV infection. Virology, 365 (1), 20-33 PMID: 17451770 

Pillai, S., Nguyen, J., Johnson, J., Haura, E., Coppola, D., & Chellappan, S. (2015). Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis Nature Communications, 6 DOI: 10.1038/ncomms10072 

Richter B, Sliter DA, Herhaus L, Stolz A, Wang C, Beli P, Zaffagnini G, Wild P, Martens S, Wagner SA, Youle RJ, & Dikic I (2016). Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 113 (15), 4039-44 PMID: 27035970 

Thurston, T., Ryzhakov, G., Bloor, S., von Muhlinen, N., & Randow, F. (2009). The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria Nature Immunology, 10 (11), 1215-1221 DOI: 10.1038/ni.1800