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Coronavirus Biology and Pathogenesis
Molecular Biology of Coronaviruses
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SARS in the Context of Emerging Infectious Threats SARS in the Context of Emerging Infectious Threats
Coronavirus Biology and Pathogenesis
Molecular Biology of Coronaviruses

Paul Masters, Wadsworth Center, NY State Dept. of Health, Albany
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Highlights

Virion Characteristics
• Coronaviruses are enveloped, positive-strand RNA viruses.
• Pleomorphic virions of 80-120 nm in diameter have a punched-in spherical appearance by negative-stained EM.
• There are characteristic 20nm surface projections on the envelope.
• The nucleocapsid is helically-symmetrical, unique among positive-strand viruses.
• Four basic structural proteins are encoded by the virus genome: spike (S), membrane (M), envelope (E), and nucleocapsid (N).

Infection and Disease Features
• Coronaviruses generally cause respiratory, enteric, and neurologic diseases.
• Infections are highly species-specific.
• Coronaviruses have been grouped by serological cross-reactivity into three groups; by genomic sequence homology; SARS is the prototype member of a fourth group.

Spike Protein
• S protein is a 150 kDa, type I membrane protein.
• Has a large N-terminal ectodomain, that is extensively N-linked glycosylated; a transmembrane domain; and short COOH-terminal endodomain.
• Oligomerizes into dimers or trimers that form the spikes.
• Binding to a receptor triggers fusing between viral envelope and a cellular membrane (plasma or endosomal) and internalization of the nucleocapsid into the cytoplasm.
• Can induce fusion of adjacent cells to form syncytia.
• Is the principle viral antigen, eliciting neutralizing antibody from the host.

Membrane Protein
• M protein is a 25 kDa, triple membrane-spanning protein, with a short N-terminal ectodomain that is either N- or O-glycosylated, and a large COOH-terminal tail that interacts with the viral nucleocapsid.
• Most abundant viral protein and a major determinant of virion morphogenesis.
• Selects S protein and the genome for incorporation into virions during viral assembly.

Envelope Protein
• E protein is an 8-10 kDa protein present in tiny stoichiometric quantities.
• Determines the site of viral budding, either ERGIC or Golgi.
• Expression of only M and E proteins leads to formation of virus-like particles and export from cells.

Nucleocapsid Protein
• N protein is a 50 kDa protein with overall positive charge.
• The protein is overall basic, but the COOH-terminus is acidic.
• Central region binds to RNA; COOH-terminus binds to M protein during assembly.
• N may serve as a translational enhancer.

The RNA Genome
• In isolation, the genome is infectious.
• Like a typical eukaryotic messenger RNA, it has a 5' cap and 3' polyadenylate tail.
• Ranges in size from 26–32 kb in length, among the largest known mature RNAs.
• Contains multiple open reading frames.
• First two-thirds of the genome contains a large 20–22 kb gene, translated via ribosomal frame-shifting into an 800 kDa polyprotein.
• The large polyprotein processes itself into 15 or 16 polypeptides that serve as a factory to replicate and transcribe the genome.
• RNA-dependent RNA polymerase and an RNA helicase are also encoded.
• There is a high rate of RNA-RNA recombination by a template-switching mechanism.

Group-Specific Nonstructural Proteins
• Genes are interspersed among structural protein genes, in the distal portion of the genome.
• Group II hemagglutinin esterase, the only nonstructural protein with a known function, provides an extra glycoprotein on the viral surface.
• Many nonstructural proteins appear to be nonessential, not expressed, or have no role in pathogenesis.

Coronavirus Life Cycle
• Binding to host cell receptors and fusion result in deposition of nucleocapsid and the genome into host cytoplasm.
• Host ribosome translates RNA-dependent RNA polymerase, which then makes a negative-strand copy of the genome and subgenomic mRNAs.
• mRNAs form a 3'-nested set, each with a 70–100 b leader sequence fused to an internal point on the genome, and a negative strand counterpart; these are translated into viral structural proteins.
• S, E, and M go to the ER and end up via the default secretory pathway in the ERGIC or Golgi.
• N protein and progeny genomes assemble into the nucleocapsid in the cytoplasm and then bud into the budding compartment, forming virions.
• One hundred to 1000 viruses are released via smooth-walled vesicles.

Reverse Genetics with Coronavirus Genome
• The first method, common for all positive-strand RNA viruses, is to produce a full-length cDNA copy that can be cloned into a transcription vector with a mutation of interest; RNA is transcribed from the cDNA and transfected into host cells to produce mutant viruses.
• This strategy has not been possible for coronaviruses until very recently because cDNA clones of many parts of the huge coronavirus genomes are highly unstable; particularly ingenious methods have been developed by the laboratory groups of Enjuanes, Baric, and Siddell to overcome this instability.
• This is the only method to do reverse-genetic on the large gene 1.
• A second method uses recombination with synthetic RNAs to construct site-directed mutations that are recovered by selecting against a thermolabile deletion mutant parent virus.
• Using this second method, Masters' and Rottier's groups have created an fMHV virus from MHV with a spike protein from feline infectious peritonitis virus; this mouse virus can only grow in feline cells and recombinants pick up a restored mouse S protein with a target mutation, allowing them to grow again in mouse cells.

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