Showing papers by "Alexander E. Gorbalenya published in 2003"

Mechanisms and enzymes involved in SARS coronavirus genome expression.

Abstract: A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and post-translational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries.

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae

Abstract: The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22–25% and 21–25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63–71% and 59–67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs

Abstract: Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CLpro]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CLpro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CLpro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827LVTHE↓VRTGN2836. The trans-processing activity of the purified recombinant 3CLpro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441KVNHE↓LYHVA6450, in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CLpro. The study revealed that the GAV and potyvirus 3CLpros possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CLpro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CLpros but in common with coronavirus 3CLpros, the GAV 3CLpro employs a Cys2968-His2879 catalytic dyad. The properties of the GAV 3CLpro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.