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.