ZIKV-infected Cells Research Articles

Selective Activation of Type II Interferon Signaling by Zika Virus NS5 Protein.

Severe complications of Zika virus (ZIKV) infection might be caused by inflammation, but how ZIKV induces proinflammatory cytokines is not understood. In this study, we show opposite regulatory effects of the ZIKV NS5 protein on interferon (IFN) signaling. Whereas ZIKV and its NS5 protein were potent suppressors of type I and type III IFN signaling, they were found to activate type II IFN signaling. Inversely, IFN-γ augmented ZIKV replication. NS5 interacted with STAT2 and targeted it for ubiquitination and degradation, but it had no influence on STAT1 stability or nuclear translocation. The recruitment of STAT1-STAT2-IRF9 to IFN-β-stimulated genes was compromised when NS5 was expressed. Concurrently, the formation of STAT1-STAT1 homodimers and their recruitment to IFN-γ-stimulated genes, such as the gene encoding the proinflammatory cytokine CXCL10, were augmented. Silencing the expression of an IFN-γ receptor subunit or treatment of ZIKV-infected cells with a JAK2 inhibitor suppressed viral replication and viral induction of IFN-γ-stimulated genes. Taken together, our findings provide a new mechanism by which the ZIKV NS5 protein differentially regulates IFN signaling to facilitate viral replication and cause diseases. This activity might be shared by a group of viral IFN modulators.IMPORTANCE Mammalian cells produce three types of interferons to combat viral infection and to control host immune responses. To replicate and cause diseases, pathogenic viruses have developed different strategies to defeat the action of host interferons. Many viral proteins, including the Zika virus (ZIKV) NS5 protein, are known to be able to suppress the antiviral property of type I and type III interferons. Here we further show that the ZIKV NS5 protein can also boost the activity of type II interferon to induce cellular proteins that promote inflammation. This is mediated by the differential effect of the ZIKV NS5 protein on a pair of cellular transcription factors, STAT1 and STAT2. NS5 induces the degradation of STAT2 but promotes the formation of STAT1-STAT1 protein complexes, which activate genes controlled by type II interferon. A drug that specifically inhibits the IFN-γ receptor or STAT1 shows an anti-ZIKV effect and might also have anti-inflammatory activity.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

Zika virus infection of adult and fetal STAT2 knock-out hamsters

Zika virus (ZIKV) infection was investigated in adult and fetal STAT2 knock-out (KO) hamsters. Subcutaneous injection of ZIKV of adults resulted in morbidity, mortality, and infection of the uterus, placenta, brain, spinal cord, and testicles, thus providing an opportunity to evaluate congenital ZIKV infection in a second rodent species besides mice. ZIKV-infected cells with morphologies of Sertoli cells and spermatogonia were observed in the testes, which may have implications for sexual transmission and male sterility. Neonates exposed as fetuses to ZIKV at 8 days post-coitus were not smaller than controls. Nevertheless, infectious virus and ZIKV RNA was detected in some, but not all, placentas and fetal brains of KO hamsters. STAT2 KO hamsters may be useful for addressing sexual transmission, pathogenesis, routes of fetal infection, and neurological disease outcomes, and may also be used in antiviral or vaccine studies to identify intervention strategies.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

The impact of Zika virus infection on human neuroblastoma (SH-SY5Y) cell line.

An increase in Zika virus (ZIKV) epidemic during the last decade has become a major global concern as the virus affects both newborns and adult humans. Earlier studies have shown the impact of ZIKV infection in developing human foetus. However, effective in vitro model of target cells for studying the ZIKV infection in adult human neurons is not available. This study aimed to establish the use of human neuroblastoma cell line (SH-SY5Y) for studying an infection of ZIKV in vitro. ZIKV growth kinetics, viral toxicity, and SH-SY5Y cell vialibity were determined after ZIKV infection in SH-SY5Y cells in vitro. ZIKV-infected SH-SY5Y cells were morphologically analysed and compared with nonhuman primate Vero cells. Furthermore, the susceptibility of SH-SY5Y cells to ZIKV infection was also determined. The results showed that ZIKV efficiently infects SH-SY5Y cell lines in vitro. Gradual changes of several cellular homeostasis parameters including cell viability, cytotoxicity, and cell morphology were observed in ZIKVinfected SH-SY5Y cells when compared to mock-treated or non-human primate cells. Interestingly, ZIKV particles were detected in the nucleoplasmic compartment of the infected SH-SY5Y cells. The results suggest that ZIKV particle can be detected in the nucleoplasmic compartment of the infected SH-SY5Y cells beside the known viral replicating cytoplasmic area. Hence, SH-SY5Y cells can be used as an in vitro adult human neuronal cell-based model, for further elucidating the ZIKV biology, and highlight other possible significance of Zika virus distribution through nuclear localization, which may correlate to the neuropathological defects in ZIKV-infected adult humans.

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models.

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.