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Journal of Virology, June 2000, p. 5213-5223, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Leonie C. van Dinten,1 Hans van Tol,1 Alexander E. Gorbalenya,2 and Eric J. Snijder1,*

Department of Virology, Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands,1 and Advanced Biomedical Computing Center, SAIC, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland 21702-12012

Received 10 January 2000/Accepted 9 March 2000

Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429right-arrowPro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429right-arrowPro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of "hinge spacer," which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.

* Corresponding author. Mailing address: Department of Virology, Center for Infectious Diseases, Leiden, University Medical Center, LUMC P4-26, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: e.j.snijder{at}lumc.nl.

Journal of Virology, June 2000, p. 5213-5223, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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