Glucose-regulated protein 78 interacts with zika virus envelope protein and contributes to a productive infection

"Zika virus (ZIKV; Flaviviridae) is a mosquito-borne flavivirus shown to cause fetal abnormalities collectively known as congenital Zika syndrome and Guillain-Barré syndrome in recent outbreaks. Currently, there is no specific treatment or vaccine available, and more effort is needed to identify cellular factors in the viral life cycle. Here, we investigated interactors of ZIKV envelope (E) protein by combining protein pull-down with mass spectrometry. We found that E interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78). Although other flaviviruses are known to co-opt ER resident proteins, including GRP78, to enhance viral infectivity, the role ER proteins play during the ZIKV life cycle is yet to be elucidated. We showed that GRP78 levels increased during ZIKV infection and localised to sites coincident with ZIKV E staining. Depletion of GRP78 using specific siRNAs significantly reduced reporter-virus luciferase readings, viral protein synthesis, and viral titres. Additionally, GRP78 depletion reduced the ability of ZIKV to disrupt host cell translation and altered the localisation of viral replication factories, though there was no effect on viral RNA synthesis. In summary, we showed GRP78 is a vital host-factor during ZIKV infection, which may be involved in the coordination of viral replication factories. © 2020 by the authors.

Glucose-regulated protein 78 interacts with zika virus envelope protein and contributes to a productive infection

Zika virus (ZIKV; Flaviviridae) is a mosquito-borne flavivirus shown to cause fetal abnormalities collectively known as congenital Zika syndrome and Guillain-Barré syndrome in recent outbreaks. Currently, there is no specific treatment or vaccine available, and more effort is needed to identify cellular factors in the viral life cycle. Here, we investigated interactors of ZIKV envelope (E) protein by combining protein pull-down with mass spectrometry. We found that E interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78). Although other flaviviruses are known to co-opt ER resident proteins, including GRP78, to enhance viral infectivity, the role ER proteins play during the ZIKV life cycle is yet to be elucidated. We showed that GRP78 levels increased during ZIKV infection and localised to sites coincident with ZIKV E staining. Depletion of GRP78 using specific siRNAs significantly reduced reporter-virus luciferase readings, viral protein synthesis, and viral titres. Additionally, GRP78 depletion reduced the ability of ZIKV to disrupt host cell translation and altered the localisation of viral replication factories, though there was no effect on viral RNA synthesis. In summary, we showed GRP78 is a vital host-factor during ZIKV infection, which may be involved in the coordination of viral replication factories. © 2020 by the authors

Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG

Purification of AdhE-D2-iLOV (P3) is easy to follow due to the distinctive yellow-green colour of the iLOV tagged protein, (A).

<p>Purified AdhE-D2 lacking the iLOV domain is colorless, (B). Purified P3 shows fluorescence under ultraviolet light, (C). Purified AdhE-D2 lacking the iLOV domain viewed under uv light (D). The iLOV domain can be readily cleaved from purified proteins using C3 protease, (E). Lane M: Markers, Lane 1: AdhE-D2-iLOV, indicated by arrow, Lane 2: AdhE-D2, iLOV and 3C protease indicated by arrows. The iLOV-His₈ tag has been cleaved off, and the size of AdhE-D2 has been reduced by approximately 16 kDa.</p

Western blot using an anti-iLOV antibody shows expression of the full-length AdhED2-iLOV-fusion protein (“P3”, A).

<p>Expression of AdhED2-iLOV-fusion protein (“P3”) over time corresponds with fluorescence, (B). AdhED2-iLOV was expressed in <i>E. coli</i> C41 cells and the level of fluorescence monitored. The arrow indicates addition of 1 mM IPTG to induce expression.</p

Heat-map of iLOV fluorescence for the 10 proteins analysed in this study.

<p>Each protein-iLOV fusion was cultured in four different media (LB, TB, 2YT and M9), three different cell lines (C41, pLysS, Rosetta) and at two different temperatures (25°C and 37°C). Peak fluorescence was determined during the growth curve. Fluorescence data were normalized from the absolute fluorescence values to a scale of 0 to 1. The area of interest was highlighted, and a “surface contour” representation was selected.</p

Microinjection of wild-type EspGΔ42 (A), EspG-iLOV (B), and buffer control (C), into cultured NRK cells.

<p>Visualized is the Golgi protein GM130, cascade blue injection dye, and merge as indicated in the vertical columns. Scale bar represents 50 µm.</p

The pET-iLOV plasmid with key features highlighted including the iLOV domain (green) and key restriction sites for cloning genes of interest.

<p>The pET-iLOV plasmid with key features highlighted including the iLOV domain (green) and key restriction sites for cloning genes of interest.</p