The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers

D.Fernando Estrada,Daniel M Boudreaux,Dalian Zhong,Stephen C St Jeor,Roberto N De Guzman

doi:10.1074/jbc.m808081200

D.Fernando Estrada, Daniel M Boudreaux + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m808081200

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Abstract

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.

Highlights

Many viruses in the family Bunyaviridae, which consists of five genera (Hantavirus, Orthobunyavirus, Nairovirus, Phlebovirus, and Tospovirus), cause emerging zoonotic infections in humans (1)
It was suggested that the cytoplasmic tails of the viral glycoproteins might bind the viral ribonucleoprotein (6)
Recent results have shown that the G1 tail binds the viral ribonucleoprotein in phlebovirus (7) and is required for packaging the genome in orthobunyavirus (8)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cysteine/histidinerich region (residues 543–599) of the G1 tail of the Andes virus (strain 23) and Prospect Hill virus was subcloned into pET-21a (Novagen) as a C-terminal fusion to a His6-tagged GB1 domain separated by a TEV protease cleavage site. The fusion protein was overexpressed in soluble form in E. coli, purified under native conditions, and digested with TEV protease to obtain the G1 zinc finger domain. Andes virus G1 zinc finger showed a folded ␣-helical domain with local minima at 209 and 222 nm (Fig. 2). NMR data were used to confirm the requirement for Zn2ϩ coordination on the proper folding of the zinc finger domain. The Andes virus and the Prospect Hill virus zinc-binding domains purified under native conditions showed well dispersed and sharp peaks in their two-dimensional 1H-15N. In the presence of excess EDTA, the HSQC spectrum of the Prospect Hill virus showed a collapse of the backbone and side chain amide peaks (supplemental Fig. S1), which indicated that the protein was unfolded. Complete backusing PROCHECK (38), and the graphics were generated using Pymol (39)

RESULTS

Disallowed regions

DISCUSSION