Mouse hepatitis virus

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Mouse hepatitis virus is a virus of the family Coronaviridae, genus coronavirus. Also known as Murine Hepatitis Virus, it is one of the best characterised coronaviruses and belongs to serogroup II. As well as hepatitis, MHV can cause respiratory, and neurological infections. Murine Hepatitis is widely seen a model for human coronaviruses, such as SARS.

1 Structure
2 Genome
3 Transcription, translation and packaging
4 Replicase proteins
5 References

[edit] Structure

The enveloped virions are approximately 120-160nm in diameter, containing a nucleocapsid consisting of the viral genome associated with the nucleocapsid phosphoprotein (N). The N protein may also have a role in RNA replication.

The helical nucleocapsid is contained within an internal core, which is spherical and composed of M (membrane) and possibly N protein.

The nucleocapsid core is surrounded by a virus membrane and the surface of all coronavirus virions have long spikes of S (spike) protein oligomers, which give the characteristic crown (in Latin, corona) morphology. Some group II coronavirus species, such as MHV, have a shorter HE (haemaglutinin-esterase) spike as well.

The envelope also contains the M (membrane) glycoprotein, which spans the envelope 3 times and also interacts with the nucleocapsid, and the E (envelope) glycoprotein.

[edit] Genome

MHV has a large, RNA genome of approximately 31kb. Gene 1 is 22kb in size and encodes the replicase polyproteins. The remaining genes encode the structural proteins, as well as some non-structural accessory proteins. The non-structural proteins vary widely depending on the species. The spike glycoprotein (S), envelope glycoprotein (E), membrane glycoprotein (M) and nucleocapsid phosphoprotein (N) are present in all coronaviruses. In the serogroup II viruses that contain it, the haemagluttinin esterase glycoprotein (HE) gene is positioned after the replicase gene, before the gene for the spike protein.

[edit] Transcription, translation and packaging

The first gene is translated directly from the genomic RNA and is the replicase gene, which encodes proteins that form a replication transcription complex (RTC), which will be discussed later. Once the RTC has been assembled, 6-8 subgenomic mRNAs are produced. The subgenomic mRNAs are positive-sense and have a 5' cap and a 5' leader sequence identical to the genomic RNA leader sequence. The leader sequence is 65-98 nucleotides in length. They are also 3’ coterminal nested with the genome (have an identical 3’ terminal sequence with to the genome). This indicates that there must be a discontinuous step in the transcription of the subgenomic mRNAs.

In the genomic RNA, between each gene, is a transcription regulation sequence (TRS), which contain a consensus sequence, (body TRS). A TRS is also found near the 3' end of the leader sequence, (leader TRS). TRS are also present in the subgenomic mRNAs, with the leader TRS marking where the body of the mRNA joins the leader sequence.

Models of transcription of subgenomic mRNA differ primarily in when the discontinuous step takes place and the direction in which the RNA is transcribed. The generally accepted model suggests that subgenomic negative strand RNAs are produced from the genomic RNA template.

The positive sense subgenomic mRNAs are then produced using the negative stranded subgenomic RNAs as a template. The discontinous transcription step takes place during the minus strand synthesis and this involves the base pairing of the body TRS on the subgenomic minus-strand RNA with the leader TRS of the genomic RNA. This leads to the production of an "anti-leader" sequence at the 3' end of the negative-strand subgenomic RNA, which when transcribed becomes the 5' leader sequence in subgenomic mRNAs.

The TRSs are essential to the effective transcription of subgenomic mRNAs, however the consensus sequence on its own is not sufficient to ensure transcription, indicating that other structures or sequences may be important. Interactions between the TRS regions of the RNA are important, but RNA-RNA interactions are not the only interactions involved. It has been suggested that protein-RNA and protein-protein interactions may also be involved.

The 5’ and 3’ untranslated regions (UTR) of the genome may also have roles in regulating the transcription and packaging of viral RNA, as well as translation.

The 3’ UTR has been shown to be essential for the replication of coronaviruses. Studies using group 2 coronaviruses, MHV and BoCV, have found that alterations in two secondary structures, the bulged loop and the pseudoknot, prevents replication of the virus. 5’ UTR secondary structures have also been indicated to contain cis-acting elements.

Both replication and transcription takes place on intracellular double membranes, however it is still unclear exactly which membranes are the site of replication and transcription. While it has been shown that MHV infection induces double membrane vesicle (DMV) formation and these act as the site of replication and transcription, it has also been shown that proteins involved with RNA synthesis localise to late endosomes and lysosomes (van der Meer et al, 1999) and that replicase complexes localise to the ERGIC (Endoplasmic Reticulum-Golgi intermediate compartment) at late time of infection.

The production of subgenomic mRNAs allows each gene to be expressed. Subgenomic mRNAs are polycistronic, but only the first open reading frame (ORF) (the ORF at the 5' end) is translated, hence only one protein is produced from each subgenomic mRNA, via the cap-dependent mechanism. There are, however, exceptions. For example, the envelope (E) protein is translated from mRNA5, despite not being the first ORF in this subgenomic mRNA and ORF1b is translated via a -1 ribosomal frameshift.

As well as the production of subgenomic mRNAs for translation into proteins, genomic RNA must also be produced for packaging into virions. Subgenomic mRNA 1, is identical to the genomic RNA and acts as an mRNA for translation of ORF1a and 1b, and is also packaged into virions.

Coronaviruses show a high frequency of recombination, when compared to other RNA viruses. MHV has a particularly high frequency of recombination (Lai, 1992). Makino et al. (1985) suggested that recombination may due to the discontinuous mechanism of RNA transcription. As the polymerase must dissociate from the template during discontinous transcription, it may re-associate at an homologous site on a different template.

Once the genomic RNA has been replicated, it forms the nucleocapsid by binding with the N protein, via a packaging signal in the RNA. The M protein then interacts with the nucleocapsid. It has been seen that M protein is able to directly interact with RNA, in the absence of the N protein and suggested that the M protein causes a conformational change in N, via this interaction, which allows the M protein to interact directly with the N protein. The M protein is essential for virion packaging, as is the E protein. The M protein incorporates HE and S into the virion and is involved in determining the site of budding, although other factors are also involved in this. The E protein helps development of virion morphology, by inducing curvature in the membrane. The S protein is not essential for virion formation.

[edit] Replicase proteins

In all coronaviruses, the replicase gene consists of two overlapping open reading frames: ORF1a and ORF1b. In MHV ORF1a can be translated either singly to produce polyprotein 1a (pp1a), which is 496.6kDa, or together with ORF1b, which requires a -1 ribosomal frame shift, to produce form polyprotein 1ab (pp1ab) of 802.8kDa. These polyproteins are cleaved by viral proteinases; papain-like proteinases PL1pro and PL2pro and 3C-like proteinase 3CLpro, forming 16 non-structural proteins, nsp1-16. Nsp1 - 10 are cleaved from ppla and pp1ab, nsp11 is only cleaved from pp1a, and nsp12-16 are cleaved from pp1ab. The proteins associate to form a replication-transcription complex (RTC).

Only a few of the nsps have confirmed functions. Nsp1 and nsp2 have not currently had any functions confirmed. However, it is known that nsp1 is the first of the non-structural protein cleaved from the amino-terminal end of the polyprotein and is important in several stages of the viral life cycle. In contrast nsp2, however, has been found to be non-essential for the replication of MHV and the replication-transcription complex has been seen to form in the absence of nsp2. Cleavage of nsp1 from nsp2 is not essential for efficient virus replication, however cleavage does result in more efficient virus replication, i.e. Increased viral RNA synthesis and viral growth, which may indicate a role for these proteins.

Nsp3 contains the papain-like proteinases; PL1 and PL2. PL1 is responsible for the proteolytic cleavage between nsp1 and nsp2, this is cleavage site 1 (CS1). It is also responsible for cleavage between nsp2 and nsp3: cleavage site 2 (CS2). PL2 is responsible for cleavage between nsp3 and nsp4: cleavage site 3 (CS3).

Other than PLP1 and PLP2 proteinases, nsp3 contains a putative transmembrane domain, TM1 and also an adenosine diphosphate ribose phosphatase (ADRP) domain which is involved in RNA processing. A cyclic phosphodiesterase (CPD) is also found in serogroup II coronaviruses, indicating that it does not have an essential function, and is thought to be involved in the same pathway of RNA processing as ADRP.

The remaining nsps are cleaved by the 3C-like proteinase (3CL), encoded in nsp5. The 3C-like proteinase is so named as it is similar to the picornavirus 3C proteinase. Either side of nsp4, in nsp3 and nsp5, are predicted membrane domains (TM2 and TM3), which are thought to anchor the replication transcription complex to the membrane, where replication takes place.

In common with other RNA viruses, MHV contains a putative RNA dependent RNA polymerase, which is found in nsp12. An RNA helicase is found in nsp13, along with a Zn binding domain.

Enzymes mapped to nsp14-nsp16 have been indicated by biochemical studies. A 3’ – 5’ exonuclease (ExoN) has been mapped to nsp14, a polyU-specific endoribonuclease (XendoU) has been mapped to nsp15 and a putative S-adenosylmethionine-dependent ribose 2’-O-methyltransferase (2’-O-MT) has been mapped to nsp16. The functions of these enzymes are all associated with RNA processing. The arrangement of these proteins in neighbouring nsps, as well as evidence that in serogroup III coronavirus, Infectious Bronchitis Virus (IBV), these 3 protein domains comprise a processing intermediate, may indicate that these domains are involved in the same pathway of RNA processing. Research has also shown that RNA which has been methylated by the 2’-O- methyltrasnsferase are resistant to processing by NendoU.

[edit] References

University of Illinois entry (PDF)
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