Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics

Savidis, George; McDougall, William M.; Meraner, Paul; Perreira, Jill M.; Portmann, Jocelyn M.; Trincucci, Gaia; John, Sinu P.; Aker, Aaron M.; Renzette, Nicholas; Robbins, Douglas R.; Guo, Zhiru; Green, Sharone; Kowalik, Timothy F.; Brass, Abraham L.

doi:10.1016/j.celrep.2016.06.028

Cited by 319 publications

(357 citation statements)

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“…Recent CRISPR-Cas9-based screenings have confirmed the importance of these groups of host proteins, most of which are directly associated with the ER function (47,48). Among these genes, SPCS1 and SPCS3 are signal peptidases which convert secretory and some membrane proteins to their mature forms by cleaving their signal peptides from their N termini; SEC61B and SEC63 are part of the SEC61 complex, which mediates protein translocation across the ER; STT3A is the catalytic subunit of the N-oligosaccharyltransferase (OST) complex, which catalyzes N-linked glycosylation; and SSR3 is a glycosylated ER membrane receptor associated with protein translocation across the ER membrane.…”

Section: Discussionmentioning

confidence: 99%

“…PKI 14-22 does not interfere with host translation machinery to inhibit ZIKV replication. By screening genes essential for ZIKV replication using the clustered regularly interspaced short palindromic repeat (CRISPR) technology, previous studies have identified several host genes that play vital roles in regulating ZIKV replication (47,48). Most of these genes are endoplasmic reticulum (ER) associated and functionally related to protein translation and modification.…”

Section: Pki 14-22 Inhibits Zikv Replication By Inhibiting the Pka Pamentioning

confidence: 99%

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Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22

Cheng

Silva

Huang

et al. 2018

J Virol

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The recent outbreak of Zika virus (ZIKV), a reemerging flavivirus, and its associated neurological disorders, such as Guillain-Barré (GB) syndrome and microcephaly, have generated an urgent need to develop effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors of ZIKV replication. Because several pathways, including the AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors and agonists of these pathways for their effects on ZIKV replication. We identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both the African and Asian/American lineages with a high efficiency and minimal cytotoxicity. While ZIKV infection does not induce PKA activation, endogenous PKA activity is essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited another unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the postentry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication which could be further explored for future therapeutic application.IMPORTANCE There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a reemerging flavivirus associated with neurological disorders, including Guillain-Barré (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. We show that PKI effectively suppresses the replication of all ZIKV strains tested with minimal cytotoxicity to human endothelial cells and astrocytes, two key cell types for ZIKV infection. Furthermore, we show that PKI inhibits ZIKV negative-sense RNA synthesis and viral protein translation. This study has identified a potent inhibitor of ZIKV infection which could be further explored for future therapeutic application.

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Section: Discussionmentioning

confidence: 99%

Section: Pki 14-22 Inhibits Zikv Replication By Inhibiting the Pka Pamentioning

confidence: 99%

Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22

Cheng

Silva

Huang

et al. 2018

J Virol

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“…Whereas Gas6 binds to all three TAM family members with high affinity, protein S binds to TYRO3 and MERTK, but not to AXL (17). The TAM receptor AXL was recently shown to support ZIKV infection of human foreskin fibroblasts (12), and its expression was noted in the brain and neuroprogenitor cells (18)(19)(20)(21). However, its deletion had no effect on ZIKV infection of induced pluripotent human stem cell-derived neuroprogenitor cells or cerebral organoids (22) or on virus accumulation of the eye, brain, or testis in Axl −/− mice (23,24).…”

mentioning

confidence: 99%

AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses

Richard

Shim

Kwon

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

184

159

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Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXLdependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.Zika virus | Flaviviruses | AXL | placental barrier | fetal endothelial cell Z ika (ZIKV), West Nile (WNV), and dengue (DENV) viruses are closely related, and belong to the Flavivirus genus in the Flaviviridae family. Although a causal relation between ZIKV and microcephaly has been established by human and animal studies (1-7), it remains unclear why only ZIKV, but not other pathogenic flaviviruses, causes congenital diseases. Although WNV is known to infect neuronal cells and results in encephalitis (8), it does not cause microcephaly. DENV is not generally neurotropic and is not linked to congenital defects.To reach the fetal brain, a virus must be transported from the maternal to the fetal circulation, which necessitates crossing of the placental barrier. In the placenta, fetal blood in capillaries is separated from maternal blood by placental barrier cells, namely trophoblasts and fetal endothelial cells. Recent studies indicate that the placenta and its barrier cells are infected by ZIKV, and fetal brain lesions develop in mice, pigtail macaques, and humans (1-6, 9). However, it remains unclear why only ZIKV, and not other neurotropic flaviviruses, results in microcephaly and other congenital disorders.Although bona fide entry receptors for flaviviruses remain unknown, many cell surface-expressed molecules contribute to infection, including C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related protein (L-SIGN) (10, 11) and phosphatidylserine (PS) receptors (12-15) . PS receptors, which serve as entry cofactors for flaviviruses, include members of the TIM (T-cell Ig mucin) family and the TAM (TYRO3, AXL, and MERTK) family. TIMfamily receptors bind PS directly (14, 15), whereas TAM-family members bind PS indirectly, through the soluble intermediates Gas6 (growth arrest-specific 6) and protein S present in serum and other bodily fluids (16, 17). Whereas Gas6 binds to all th...

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“…This is largely due to the varied efficiency of multiple steps of entry leading to productive infection. Whereas multiple host factors are involved in late post-fusion steps of virus entry (1)(2)(3)(4)(5)(6)(7)(8)(9), the effects of intrinsic, cell type-dependent factors and extrinsic factors on viral fusion are poorly characterized. After the initial interaction of viruses with cellular receptors or attachment factors, low endosomal pH is required to trigger fusion-inducing conformational changes in most viral proteins (reviewed in Refs.…”

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confidence: 99%

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

Desai

Marin

Mason

et al. 2017

Journal of Biological Chemistry

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Edited by Thomas SöllnerEnveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.Infectivity of enveloped viruses often varies depending on the cell type, even for cells expressing comparable levels of cognate receptors. This is largely due to the varied efficiency of multiple steps of entry leading to productive infection. Whereas multiple host factors are involved in late post-fusion steps of virus entry (1-9), the effects of intrinsic, cell type-dependent factors and extrinsic factors on viral fusion are poorly characterized. After the initial interaction of viruses with cellular receptors or attachment factors, low endosomal pH is required to trigger fusion-inducing conformational changes in most viral proteins (reviewed in Refs. 10 and 11). The low pH requirement of virus entry could also stem from the need for optimal milieu (e.g. endosomal protease activity) for priming the viral glycoproteins for the fusion reaction (10). It is thus likely that cell type-dependent regulation of endosomal pH modulates the efficiency and kinetics of virus fusion. To date, however, only a few studies have directly examined the link between the pH in virus-carrying endosomes and the efficiency/kinetics of subsequent viral fusion (12-15).Endosome-resident lipids and proteins have been implicated in the completion of virus fusion and/or the nucleocapsid release into the cytoplasm (8, 16 -22)...

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Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics

Cited by 319 publications

References 50 publications

Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22

Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22

AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses

pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

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