Structure and genetic analysis of the arterivirus nonstructural protein 7alpha

Abstract

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7β, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.

Fig. 1.

The NMR structure of EAV nsp7α. (A) Cα trace of the 20 lowest-energy nsp7α solution structures superimposed for residues Asp11 to Glu122. (B) A cartoon representation in the same orientation as panel A. α-Helices are colored blue, β-strands magenta, and β-turns pink. The N and C termini are labeled, as are the surface residues referred to in the text discussing the mutagenesis results. The H-bonded residues Arg24 and Asp81 are shown in stick representation and colored according to atom type (C, green; N, blue; O, red). (C) A cartoon representation showing the hydrophobic residues that form the core of the fold. The residues are shown in stick representation and as semitransparent spheres with a radius equal to the van der Waals radius of the atoms. For clarity, the residues are divided by color into four hydrophobic patches, namely, red (Phe14, Leu17, Leu48, Leu52, Leu64, and Val109; Leu17 is fully obscured by Leu48 and was not labeled for clarity), magenta (Val26, Leu36, Ala72, and Phe71), blue (Phe16, Val83, and Ile116), and green (Ala25, Val77, Leu101, and Val119). Panels B and C were produced with ccp4mg (14).

Fig. 2.

Surface features, conservation, and mutagenesis of EAV nsp7α. Panels A to C are in the same orientation and are related to D to F by rotation of 180° about the horizontal axis. The semitransparent surfaces in panels A and D are colored according to sequence conservation, with red least conserved and blue most conserved. Panels B and E are colored according to electrostatic potential, with red corresponding to negative charge and blue to positive charge. For comparison, panels C and F are cartoon representations using the same coloring as in Fig. 1B. (G) Alignment of amino acid sequences of arterivirus nsp7α proteins, coupled with secondary-structure information from the EAV nsp7α three-dimensional structure. The alignment is based on sequence data for EAV (RefSeq no. NC_002532), European porcine respiratory and reproductive syndrome virus (PRRSV-EU; RefSeq no. M96262.2), North American porcine respiratory and reproductive syndrome virus (PRRSV-NA; RefSeq no. NC_001961), lactate dehydrogenase elevating virus strain Plagemann (LDV-P; RefSeq no. NC_001639), and simian hemorrhagic fever virus (SHFV; Refseq no. NC_003092). The alignment was produced with ClustalW2 (8) and edited with JalView. Residues are colored according to the standard ClustalW2 color scheme to indicate conservation. The (predicted) nsp7α/nsp7β cleavage site is indicated with an arrow, and the alignment of the region surrounding the nsp7α/nsp7β junction is based on a previously published sequence comparison (20). Asterisks indicate the EAV nsp7α residues that were probed by reverse genetics in this study (Leu17, Arg24, Asn31, Arg33, Phe39, Arg62, Arg63, Lys67, Phe71, Asp81, and Gly111). Panels A to F were produced with ccp4mg (14).

Fig. 3.

Genotype and phenotype of EAV nsp7α mutants. The details of EAV nsp7α mutants engineered in this study are listed, and amino acid numbers for both the EAV pp1a/pp1ab replicase polyproteins and the 123-aa nsp7α protein described in this study are provided. BHK-21 cells transfected with synthetic full-length EAV RNA carrying these mutations were analyzed by immunofluorescence microscopy at 16 and 24 h posttransfection, and supernatants were harvested for plaque assays. Plaque assays were fixed and stained with crystal violet at 40 h postinfection, and images of a selection of mutants are shown in the lower part of the figure. wt, wild type.