key: cord-1005742-h9078ozy
authors: Frey, Teryl K
title: Chapter 501 Rubella Virus Non-Structural Protease
date: 2013-12-31
journal: Handbook of Proteolytic Enzymes
DOI: 10.1016/b978-0-12-382219-2.00495-6
sha: 332a7fc3c73d431c003caa727429b891f82586e1
doc_id: 1005742
cord_uid: h9078ozy

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 501 is Rubella Virus Non-structural Protease. Keywords: Alphavirus protease, Ca binding motif, calmodulin binding motif, catalytic Cys-His dyad, papain-like cysteine protease, regulation of virus replication, regulation of virus RNA polymerase activity, togavirus protease, virus evolution, virus-encoded protease, virus peptidase, Zn binding motif.

Rubella virus, a positive-strand RNA virus belonging to the Rubivirus genus of the Togaviridae family (reviewed in [1] ), contains two open reading frames within its genome of roughly 10 000 nucleotides: a 5 0 -proximal ORF encoding non-structural proteins involved in RNA replication (NSP-ORF) and a 3 0 -proximal ORF encoding the virion structural proteins, a capsid protein and two envelope glycoproteins (SP-ORF) (Figure 501 .1A). Whilst the proteins encoded by the SP-ORF are cleaved co-translationally by cellular signal peptidase (Chapter 778), the product of the NSP-ORF is processed by an embedded papain-family cysteine protease. The name rubella virus non-structural protease is applied to this enzyme as it is a virally encoded endopeptidase functioning in the processing of the non-structural proteins of the virus.

The product of the rubella virus NSP-ORF is 2116 amino acids in length (roughly 240 kDa but termed P200) (Figure 501 .1A). The protease domain resides roughly between residues 1000 and 1301 of the NSP-ORF. The catalytic dyad consists of Cys1152 and His1273. The protease catalyzes a single cleavage between Gly1301 and Gly1302 (28 residues from the catalytic His) producing two products: an N-terminal 150 kDa protein (termed P150) and a C-terminal 90 kDa protein (P90); the protease is thus the C-terminal domain of P150. The protease cleaves both in cis and in trans [2, 3] . Whilst no other cleavages within P200 have been detected, it is unknown if the protease cleaves any cellular proteins.

The rubella virus non-structural protease contains binding sites for both Zn 21 and Ca 21 ions. Zn binding is coordinated by four Cys residues within a C-X-X-C-(X48)-C-X-C linear context [4] (Figure 501 .1A,B). Ca binding is coordinated by a 12 amino acid linear 'EF hand' motif [5] ( Figure 501 .1A,B). Binding of both ions plays an essential structural role in protease function. Zn binding serves to shield hydrophobic residues from the solvent while Ca binding enhances the thermal stability. In fact, a mutation which abrogates Ca binding renders the protease temperature sensitive at 39 versus 35 C. Recently, it was found that the rubella virus non-structural protease binds Ca-charged calmodulin [6] (Figure 501.1A,B) . Binding is to an alpha helix adjacent to the Cys1152 catalytic residue and serves to stabilize the helix.

In the replication cycle of a positive-strand RNA virus, the genomic positive stand is first copied into a negative strand genomic equivalent that serves as the template for the synthesis of more positive strands. Positive strand RNAs are needed in stoichiometric amounts to serve as mRNAs and to be packaged into progeny virus particles in contrast to negative strands that only serve as re-usable templates. Therefore, positive-strand RNA viruses exhibit asymmetrical production of these species with positive-strands predominating over negativestrands. In the case of rubella virus, asymmetrical production appears to be mediated by the non-structural protease in the following fashion [7] : Whereas the P200 precursor catalyzes synthesis of the negative strand following its translation from the genomic RNA, the P150/ P90 complex that is produced subsequent to cleavage only synthesizes positive strands. Thus, the proteolysis both alters the catalytic activity of the replicase complex, and removes the negative-strand synthase ensuring asymmetric production of positive strands. This scheme was first worked out with the alphaviruses, the other Togavirus genus [8, 9] .

The rubella virus non-structural protease is related to a number of other viral papain-like proteases, including proteases expressed by the alphaviruses, the arteriviruses, the coronaviruses and foot-and-mouth disease virus of animals, and the potyviruses and hypovirulence-associated viruses of plants and fungi (reviewed in Chen et al. [10] ). The existence of these proteases in these diverse viruses (which belong to different families as well as infecting animals and plants) and their relatedness to cellular papain-like proteases indicates both that these viral proteases were 'captured' from cells and that this capture event occurred several times independently during evolution of these viruses. Considering the apparent independent acquisition of these proteases, it is not surprising that the function of the proteolytic cleavage mediated by these proteases differs among these diverse viruses [11] .

For a review, see Ten Dam et al. [12] . 

Molecular biology of rubella virus

The rubella virus nonstructural protease requires divalent cations for activity and functions in trans

Rubella virus nonstructural protein protease domains involved in trans-and cis-cleavage activities

A cysteine-rich metal-binding domain from rubella virus nonstructural protein is essential for viral protease activity and virus replication

Identification of a Ca 21 -binding domain in the rubella virus nonstructural protease

Calciumdependent association of calmodulin with the rubella virus nonstructural protease domain

Rubella virus RNA replication is cispreferential and synthesis of negative-and positive-strand RNAs is regulated by the processing of nonstructural protein

Polypeptide requirements for the assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus-and plus-strand RNA synthesis

Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis

Characterization of the rubella virus nonstructural protease domain and its cleavage site

Putative papain-related thiol proteases of positive-strand RNA viruses

Virus-encoded proteinases of the Togaviridae