Cytopathogenesis of ZIKV: IFTIM and viral entry/Hofbauer cells and the placenta

Zika Virus (ZIKV) is an emerging member of the Flaviviridae that in the past months spread to over 30 countries in the Americas alone that has been associated with the emergence of microcephaly, a condition associated with abnormal brain development, in Brazil and elsewhere.

Recent studies confirmed the presence of ZIKV in the amniotic fluid as well in the brain and CNS of neonates, suggesting that ZIKV can infect embryonal and foetal tissue by crossing the placental barrier. In addition, ZIKV was detected in chronic villi of the placenta in one infected mother, suggesting that ZIKV can indeed be transmitted vertically.

 

Cytopathic effects of ZIKV in cell lines derived from human neural progenitor cells and the role of HC in transmitting ZIKV in utero

Recently published studies which have been discussed 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 cell. Although these studies suggest that ZIKV can replicate in these cells and induce apoptosis in these cells -results that by large confirmed in mouse models -  one of the concerns is that those cells do not fully recapitulate foetal gene expression when compared to human neural progenitor cells (hNP) which are directly obtained from foetal brain tissue. In addition, iPSC are derived from non-neural cells and therefore need to be validated prior using them to study ZIKV pathogenesis in particular when investigating the potential consequences of ZIKV on the development of the foetal brain.

Therefore, in a recent study two foetal cell lines derived tissue at 16-19 days prof gestation were generated with cells being infected with a recent ZIKV isolate from Puerto Rico isolated in 2019 (ZIKV PRVABC59/ZIKV PR2019) at an MOI of 0.5. Following the infection with ZIKV PR2019, the viral E protein can be detected at 48 hrs p.i. by immunofluorescence in Nestrin positive with an infection rate of approximately 16% as measured by intracellular flow cytometry analysis, thus confirming previous results obtained from infected hNPC and brain organoids that ZIKV can indeed infect neuronal cells.

Concomitant with the detection of viral antigen, a significant number of infected but not non-infected neighbouring cells exhibit pyknotic cells and activated caspase-3 positive cells indicative of apoptosis, confirming previous results obtained in brain organoids, hNPC, neurospheres and in foetal mice brains thus suggesting that the induction of apoptosis in neuronal by ZIKV is a common feature of ZIKV isolates belonging to the Asian lineage.

In order to determine if the infection of neuronal cells with ZIKV PRVABC559 induces the expression and secretion of cytokines –and thus induces apoptosis of infected cells in a paracrine manner, bystander apoptosis of neighbouring cells or releases neuroprotective factors that would prevent apoptosis of neighbouring neuronal cells- the supernatant of ZIKV infected cells was analysed for the presence of a total 102 human cytokines and chemokines, including TNF-α, CCL2 and a neuroprotective cytokine, CX3CL1. In contrast to neuronal cells treated with Poly (I:C) (a mimic of dsRNA that induces the expression of cytokines), ZIKV PRVABC59 does not induce the expression of cytokines at both 24 hrs and 72 hrs p.i.. As discussed before, ZIKV infection of cell lines and brain organoids activates TLR-3 and inhibition of TLR-3 has been reported to increase ZIKV replication, thus suggesting that ZIKV inhibits antiviral signaling pathways downstream of TLR-3 albeit not completely. In line with these results, the viral NS5 protein has recently been reported to inhibit STAT2 mediated signaling by preventing the nuclear import of STAT2 in a species specific manner. Although the role of NS5 has not been studied in hNP, hNPC or brain organoids, it might be possible that ZIKV PR2019 NS5 inhibits STAT2 in these model systems in a similar way.

Similar to hNP cells, THP-1 cells also failed to induce the expression of cytokines, including Interferon-α. These results are also important when investigating the possibility of productive ZIKV replication in placental cells. As discussed before, placental cells express high levels of Interferon-λ1 (IFN-λ1), thus potentially inhibiting ZIKV replication; assuming that ZIKV prevents the induction of IFN-λ1, ZIKV might be able to replicate in placental cells albeit maybe to lower levels when compared to hNP.

The notion that ZIKV can replicate in placental cells at low levels is supported by recent findings that in syncytioblasts (differentiated cytotrophoblasts; CTB) derived from healthy donors ZIKV PR2019 persistent viral RNA can be detected up to 72 hrs p.i. concomitant with the release of infectious viral particles up to 96 hrs p.i., the latter increasing five-fold between 72 and 96 hrs p.i. The infection of CTB (as well as Hofbauer cells, see below) with ZIKV PR2019 induces an increase in IFNB1 transcripts in the absence of Interferon-β secretion indicating that ZIKV might inhibit secretion of IFN-β and/or the translation of IFNB1 mRNA. In this context it is interesting to note that both Coxsackievirus B and Poliovirus encode for viral proteins that interfere with the cellular secretory pathway and that a chimeric yellow fever/dengue virus is released in secretory vesicles suggesting that ZIKV might inhibit the secretion of IFN by diverting the cellular secretory pathway in favour of the release of viral particles. Further studies are however warranted.

The findings that ZIKV PR2019 can replicate in placental cells seem at first to contradict previous results indicating that ZIKV replication might be inhibited in placental cells due to the naturally high levels of IFN- λ1. In contrast to the most recent findings however, the previous results suggested that the supernatant of human placental cells might prevent the replication of ZIKV FSS13025 and ZIKV MR766 in permissive JEG-3 cells, whereas the current study examined if placental cells per se are permissive to ZIKV.

In contrast to hNP, the infection of Hofbauer cells (HC), M2 type macrophages that are considered to be anti-inflammatory, with ZIKV PR2019 not only supports viral replication with high viral titres detectable as early as 48 hrs p.i., but also increased secretion of IFN-α, IL-6, MCP-1 and IP-10, suggesting that ZIKV infection of HC –similar to other viruses- induces a strong antiviral response. In doing so, ZIKV infection of HC might protect uninfected bystander cells from being infected and indeed may activate and induce the maturation of monocyte derived dendritic cells, thus priming adaptive T cell response similar to DENV infected monocyte derived cells. However, infection of HC has also been implicated in contributing to the infection of embryonal cells by crossing the placental barrier and infection embryonal/foetal neuronal precursor cells.

The induction of the expression of genes encoding for pro-inflammatory cytokines and antiviral chemokines by ZIKV is preceded by the induction of the expression of retinoic acid inducible gene -1 (RIG1) like receptor (RLR) as well as downstream antiviral genes including MDA5, DDX58, DHX58, and IFIT-1/-2/-3. Interestingly, gene expression analysis of foetal radial glial cells in mouse pups infected with ZIKV SZ01 revealed a similar induction of the antiviral response, suggesting that at least in the foetal brain of immunocompetent mice ZIKV can elicit a strong antiviral response.

Figure: Expression of MDA5, RIG-1, IFIT-1, IFIT-2 and IFIT-3 is increased
in  ZIKV SZ01 infected glial cells

In conclusion, ZIKV PR2019 infected both placental cells and Hofbauer cells, albeit with different kinetics. From the current data it is not clear whether Hofbauer cells get infected first and then infect placental cells, or vice versa (which seems to be unlikely). It is important to note that HC are present in the placenta up to 18 weeks gestational age. Transplacental transmission however is not unique to ZIKV. In the past, HBV has been shown to cross the placental barrier, probably by cell-to-cell spread.  In contrast to ZIKV however most infections occur during the third trimester whereas ZIKV transmission predominantly occurs within the first two trimesters.

In any case, the infection of either of both cell types might be responsible for the transmission to the developing foetus, in particular neural progenitor cells. Infected hNP in contrast to HC and placental cells do not produce pro-inflammatory and antiviral chemokines but undergo apoptosis; interestingly, infected HC do not undergo apoptosis whilst producing both pro-inflammatory and antiviral chemokines. So far no gene expression data are available to determine if ZIKV regulates the expression of genes related to apoptosis, autophagy or the DNA damage repair.

   

ZIKV and IFTIM: inhibition of viral replication and promoting apoptosis?

IFN-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral and antibacterial response with IFTIM-1, -2 and -3 blocking the entry of a broad spectrum of RNA viruses including Influenza A virus, SARS-CoV, West Nile Virus (WNV), Dengue Virus (DENV) and a reporter virus carrying the envelope of Omsk hemorrhagic fever virus. In the case of DNA viruses, neither IFTM-1 nor IFTM-2 or IFTM-3 preventing the entry of Human Cytomegalovirus (HCMV), Human Papillomavirus (HPV) nor Adenovirus whilst African Swine Fever (ASFV) isolate Ba71V induces the expression of IFTIM-2 in Vero cells, thus preventing decapsidation of the viral particle and viral entry. In the case of ZIKV, the infection of Vero cells but not human or murine fibroblasts as well as Hela cells with ZIKV MR766 (and in the case of HeLa cells, ZIKV FSS13025) induces extensive apoptosis of infected cells 72 hrs p.i. . In ZIKV MR766 infected hNPC, IFTIM-2 is downregulated, suggesting that IFTIM-2 downregulation might contribute to ZIKV induced apoptosis via decreasing BAG-3 dependent paracrine prosurvival signaling. The importance of IFTIM for the survival of ZIKV MR766 or ZIKV FSS13025 infected HeLa cells is evident from recently published experiments showing that in HeLa cells expressing shIFTIM3 (thus downregulating the expression of both IFTIM-2 and -3) viral induced apoptosis is significantly increased at 72 hrs p.i.

Figure: ZIKV entry might be dependent of IFTIM-2/-3 and inhibited by ZIKV at late time points
p.i. 

In addition to contributing to the induction of apoptosis in infected cells, the deletion of IFTIM-3 in murine fibroblasts also increases the replication of (mouse adapted) ZIKV MR766 and which can be reversed by overexpression of IFTIM-3. Moreover, overexpression of IFTIM-3 in human A549 cells decreases the replication of both ZIKV MR766 and ZIKV FSS130025 probably by inducing antiviral signaling pathways, preventing decapsidation similar to WNV, DENV and ASFV and/or promoting the degradation of endosomes containing the viral capsid via fusion with the lysosome. From an experimental point of view, the recently described infectious clone of ZIKV might be modified to allow for live cell imaging of ZIKV entry in the absence of IFTIM-2 and -3.

It should be emphasized that this mechanism does not apply to the initial infection but only would prevent reinfection of already infected cells. Therefore, further research using a replication-defective virus or UV inactivated virus is needed to clarify the role of IFTIM in viral entry.

As described for VSV-G and EBOV in a previous posting, viral entry and subsequent targeting of viral capsid(s) to the lysosome might involve components of autophagy machinery, in particular UVRAG, thus promoting the formation of LBPA positive acidic vesicles. Although it has not been demonstrated for ZIKV, ZIKV entry might be dependent on ATG9. In this scenario, absence of ATG9 might promote the formation of LBPA positive acidic vesicles similar to Bluetongue Virus.

Figure: ZIKV capsid might localise to LBPA positive acidic vesicles in a UVRAG dependent manner

Further studies are however needed to establish if a deficiency or the expression of a mutant allele of the gene encoding for IFTIM-3 contributes to the spread of ZIKV in the Americas and increases susceptibility of foetuses to ZIKV infection or contributes to the vertical transmission of ZIKV. Data from Asia suggest the prevalence of a mutant rs12252-C allele (encoding a mutant form of IFTM-3) thus rendering individuals for increased risk of severe influenza. Whether this also contributed to the spread of ZIKV in Asia is however not clear.

Further reading

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