Selective Replication of Coronavirus Genomes That Express Nucleocapsid Protein

Abstract

The coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in coronavirus replication and transcription. We found that human coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one coronavirus structural protein, the N protein, is involved in coronavirus replication.Coronaviruses are enveloped, positive-strand RNA viruses that are mainly associated with enteric or respiratory diseases in humans, companion animals, and livestock. Coronavirus particles contain a genomic RNA of approximately 27,000 to 30,000 nucleotides and four structural proteins: the spike glycoprotein S, the membrane protein M, the small envelope protein E, and the nucleocapsid protein N (44). Three of these four proteins are embedded in the viral envelope. These are the S protein, which mediates binding of the virus particle to the target cell and the subsequent fusion of viral and cellular membranes (8), the M protein, which has a crucial role in the incorporation of the virus nucleocapsid into virus particles (28,30,31), and the E protein, which facilitates virus assembly, possibly by inducing curvature into pre-Golgi membranes, the site at which coronaviruses assemble by budding (13, 36). The fourth structural protein, N, is associated with the viral RNA genome to form a ribonucleoprotein complex (38).The coronavirus N protein has been described as a multifunctional protein displaying RNA-binding activity, proteinprotein interaction (specifically with the M protein), and the ability for self-association (22,29,30). Clearly, many of these features will reflect the structural role of the N protein in the virus particle. However, there are also some observations that suggest the N protein may have a role in viral RNA synthesis. First, several studies have provided evidence for the binding of N protein to coronaviral RNA sequences that are involved in the regulation of RNA synthesis. These sequences include the coronavirus leader sequence, transcription regulatory sequences (TRS), and sequences corresponding to the 3Ј end of coronavirus genomes (2,10,11,24,34,48). Second, it has been shown that in addition to a cytoplasmic distribution within the host cell, at least a fraction of the coronavirus N protein colocalizes with replicative proteins at the site...

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…Genome-length RNAs were prepared by in vitro transcription by using purified genomic DNA from vHCoV-inf-1 and its mutant derivatives (38). Fifteen micrograms of HCoVinf-1 RNA, HCoV-1ab-D5682A͞E5684A RNA, HCoV-1ab-D5834A RNA, HCoV-1ab-H5859A RNA, and HCoV-1ab-D5864A RNA, respectively, were used for electroporation of 4 ϫ 10 6 BHK-21 cells expressing the HCoV-229E nucleocapsid (N) protein (BHK-HCoV-N cells) (39). Seventy-two hours after transfection, poly(A) RNA was isolated, separated by agaroseformaldehyde gel electrophoresis, and transferred to nylon membrane.…”

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis

“…The generation of the SARSCoV replicon was based on the full-length cDNA clone described above and on published data showing that the N protein is required for efficient CoV replication (2,22). Accordingly, a cDNA containing the untranslated 5Ј and 3Ј ends of the Urbani strain genome, the replicase gene, and the N gene preceded by the upstream 143 nt including its natural transcription-regulating sequence was cloned as a BAC under the control of the CMV promoter by following the same approach described for the generation of the full-length cDNA clone (Fig.…”

Bronquite infecciosa das galinhas: conhecimentos atuais, cepas e vacinas no Brasil

“…O gene da polimerase consiste de duas estruturas de leitura abertas: ORF1a e ORF1b, que participam da transcrição do RNA e processam as proteinases responsáveis pela clivagem da poliproteína em produtos funcionais, sendo o gene da polimerase necessário e suficiente para a replicação e transcrição do genoma. Entretanto, a proteína do NC aumenta consideravelmente a replicação do genoma do coronavírus (SCHELLE et al, 2005), sugerindo que essa proteína possui papel importante na regulação da replicação do coronavírus. A poliproteína 1ab (pp1ab) contém uma proteinase 3C-semelhante (3CLpro) e uma proteinase papaína-semelhante (PLP), que dão origem a 15-16 proteínas não estruturais (nsps).…”