Jens Herold

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.

Poliovirus Requires a Precise 5′ End for Efficient Positive-Strand RNA Synthesis

ABSTRACT Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5′ end, which are deleted during replication to generate authentic viral genomes. We removed those 5′-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5′ ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5′ end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.

Viral Replicase Gene Products Suffice for Coronavirus Discontinuous Transcription

ABSTRACT We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5′ and 3′ ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.

Effective amplification of 20-kb DNA by reverse transcription PCR.

Abstract Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.

Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells.

The translation products of the human coronavirus (HCV) 229E open reading frames 1a and 1b, the polyproteins 1a and 1ab, are processed by virus-encoded proteinases. One of the key enzymes in this process is a chymotrypsin-like enzyme, the 3C-like proteinase. In this study we have identified an ORF 1b-encoded, 41 kDa processing product in HCV 229E-infected cells by using a monoclonal antibody with defined specificity. We show that this polypeptide is released from polyprotein 1ab by 3C-like proteinase-mediated cleavage of the peptide bonds Gln-6110/Gly-6111 and Gln-6458/Ser-6459. Also, we have investigated the subcellular localization of the 41 kDa processing product. Immunofluorescence microscopy revealed a punctate, perinuclear distribution of the 41 kDa polypeptide in infected cells and an identical subcellular localization was observed for three additional pp1ab-derived polypeptides. In contrast, the virus nucleocapsid protein showed a homogeneous cytoplasmic localization.

Molecular analysis of the human coronavirus (strain 229E) genome

The nucleotide sequence of the human coronavirus strain 229E (HCV 229E) has been determined. This article describes the organization of the virus genome, the predicted viral gene products and the mechanisms which regulate viral gene expression. This information provides a basis to investigate the biology and pathogenesis of HCV.

A Strategy for the Generation of Infectious RNAs and Autonomously Replicating RNAs Based on the HCV 229E Genome

A strategy to generate in vitro transcripts representing infectious RNAs and autonomously replicating RNAs based on the HCV 229E genome is presented. PCR-DNAs were ligated in vitro, resulting in 27 kbp and 22 kbp ligation products. These DNAs can now be transcribed in vitro and the RNAs tested for infectivity and their ability to replicate.

Characterization of the Human Coronavirus 229E (HCV 229E) Gene 1

The sequence of the HCV 229E gene 1 has been determined and compared with the homologous sequences of the murine hepatitis virus and the avian infectious bronchitis virus. The coding sequence of gene 1 is 20,273 nucleotides in length. Within this coding region are two large open reading frames, ORF 1a (4,086 codons) and ORF 1b (2,687 codons) which overlap by 40 nucleotides. In the overlapping region, the genomic RNA can be folded into a pseudoknot structure, an element which is known to mediate -1 ribosomal frame-shifting in other coronaviruses. Assuming that -1 frame-shifting occurs at the HCV sequence UUUAAAC (nucleotides 12,514-12,520), the ORF 1a - ORF 1b product is predicted to be 6,758 amino acids in length. Our sequence analysis of the HCV 229E gene 1 has revealed a high degree of similarity within the ORF 1b of HCV, MHV and IBV, whereas ORF 1a is much less conserved. Elements which are believed to be necessary for specific (e.g. frame-shifting) and general (e.g. NTP-binding/helicase) transcriptional functions have been identified. This study completes the genomic sequence of HCV 229E which is 27.27 kb long and one of the largest known RNA genomes.

Characterization of a Papain-Like Cysteine-Proteinase Encoded by Gene 1 of the Human Coronavirus HCV 229E

Expression of the coronaviral gene 1 polyproteins, pp 1a and pp 1ab, involves a series of proteolytic events that are mediated by virus-encoded proteinases similar to cellular papain-like cysteine-proteinases and the 3C-like proteinases of picornaviruses. In this study, we have characterized, in vitro, the human coronavirus HCV 229E papain-like cysteine-proteinase PCP 1. We show that PCP 1 is able to mediate cleavage of an aminoterminal polypeptide, p9, from in vitro translation products representing the aminoproximal region of pp 1a/pp 1ab. Mutagenesis studies support the prediction of Cys1054 and His1278 as the catalytic amino acids of the HCV 229E PCP 1, since mutation of these residues abolishes the proteolytic activity of the enzyme.

Long Distance Reverse-Transcription PCR

Polymerase chain reaction (PCR) has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs (1-5). We have adapted the concept of long PCR technology to reverse-transcription (RT) PCR (6). Here, we describe the parameters critical in producing RT-PCR products of up to 20 kbp. The nature of RT-PCR requires the synthesis of a cDNA by RT prior to its amplification in the PCR reaction. Thus, we focus on the three steps of RT-PCR: the preparation and requirements of the RNA template, the reverse transcription reaction, and the amplification of the cDNA by PCR.