Eric J. Snijder

The molecular biology of arteriviruses.

.The establishment of an arterivirus family acknowledgesthemanyuniquebiologicalandmolecularpropertiesofitsfourpresent members. On the other hand, the affiliation with thecoronavirusesrecognizesanintriguingancestralrelationshipatthelevelofgenomeorganizationandreplication.ThegenomesequencesofEAV,PRRSV,LDVandSHFVwereinstrumentalin uniting and reclassifying the arteriviruses (den Boon

The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the ∼30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.

De Novo Initiation of RNA Synthesis by the Arterivirus RNA-Dependent RNA Polymerase

ABSTRACT All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3′ end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3′-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

The Coronaviruslike Superfamily

Until recently, the Coronaviridae was classified as a monogeneric family of closely related viruses. However, in the past four years, it has become evident that similarities in the genome organization, replication strategies, and nucleotide sequences of coronaviruses, toroviruses, and arteriviruses, require a revision of this taxonomy. The “superfamily” concept (Strauss and Strauss, 1988; Goldbach and Wellink, 1988), which is based on evolution and phylogeny and which has already closed the gaps between other virus groups (e.g., the alpha-viruslike and picornaviruslike superfamilies), can now also be applied to a group of “coronaviruslike viruses.”

The Arterivirus Replicase

About seven years ago, the identification of arteriviruses (den Boon et al., 1991) and toroviruses (Snijder et al., 1990) as distant relatives of “traditional” coronaviruses incited a discussion on the taxonomic position of these three virus groups (Cavanagh et al., 1994). As a first result, the genus torovirus was included in the Coronaviridae family (Cavanagh et al., 1993). The taxonomic debate ended at the 1996 International Congress of Virology in Jerusalem with the establishment of the Arteriviridae family and the order of the Ni–dovirales, containing the Coronaviridae and Arteriviridae families (Cavanagh, 1997). These re–classifications acknowledged both the many unique properties of arteriviruses and coronaviruses as well as their intriguing ancestral relationship at the level of replicase genes, genome organization, and replication strategy (Snijder and Horzinek, 1993; Snijder and Spaan, 1995; Cavanagh, 1997; de Vries et al., 1997; Snijder and Meulenberg, 1998).

Proteolytic processing of the arterivirus replicase.

Arteriviruses are enveloped positive-stranded RNA viruses which belong to the so-called ‘coronaviruslike superfamily’1,2,3,4. At present, the arterivirus group is comprised of equine arteritis virus (EAV, the prototype of the group), lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV, also known as ‘Lelystad virus’), and simian haemorrhagic fever virus (SHFV). Their isometric nucleocapsid core contains a nonsegmented genome of 12.7–15.1 kb1,5,6. The morphological charac-teristics and genome size of EAV are most comparable to those of togaviruses and flaviviruses. However, the arterivirus replication strategy is similar to that of coronaviruses, which possess 25–31 kb positive-stranded RNA genomes. Among their common features are the polycistronic genome organization, the same basic gene order (5′-replicase gene-envelope protein genes-nucleocapsid protein gene-3′), and the production of a 3′-coterminal nested set of 4 to 7 subgenomic mRNAs. The 5′ part of the genomes of these viruses is occupied by two large open reading frames (ORF la and ORF lb) which encode the viral replicase1,5–12. Both ORFla and ORFlb are expressed from the genomic RNA, the latter by means of ribosomal frameshifting1,13. The ORFlb products of various members of the coronaviruslike group contain a number of homologous domains1,6,9 which indicate that the replicase genes of these viruses are evolutionarily related (Fig. 1).

Characterization of an Arterivirus Defective Interfering RNA

Recently, we have described the generation of DI-b, a natural equine arteritis virus (EAV) defective interfering (DI) RNA of 5.6 kb, and we have reported the construction of pEDI, a full-length cDNA copy of EAV DI-b RNA from which replication-competent RNA can be transcribed in vitro (Molenkamp et al., 2000a, Molenkamp et al., 2000b). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene (Fig. 1). As a result the EDI RNA contains a truncated replicase open reading frame (ORF), which we will refer to as EDI-ORF. The importance of such a translation unit in Coronavirus DI RNA propagation has been shown by a number of researchers (de Groot et al., 1992; van der Most et al.,1995; Liao and Lai, 1995).