Electron microscope image of mouse leukaemia virus-like particles containing a faulty p12 protein.

Kate Bishop : The function of the MLV p12 protein

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The MLV p12 protein is essential for both early and late stages of viral replication. We are studying the function of p12 during early, post-entry replication.

All retroviral genomes contain a gag gene that codes for the Gag polyprotein. Gag is cleaved upon viral maturation to release individual proteins, including matrix, capsid and nucleocapsid, providing the structural components of the virion. In murine leukaemia virus (MLV), Gag cleavage releases an additional protein, named p12, required for both early and late stages of the viral life cycle. We are characterising the role of p12 during early events. Viruses carrying mutations in p12 are able to reverse transcribe their genomes but cannot integrate this nascent DNA.

Using mutagenesis studies combined with microscopy and biochemical experiments, we mapped two functional domains in p12. We showed that the N-terminal motif of p12 binds directly to the capsid protein and that mutations in this motif affect the structure and stability of the viral capsid shell.

Additionally, we have demonstrated that p12 is involved in directing the viral pre-integration complex to chromatin ready for integration. We are continuing to use p12 to unravel the key processes of capsid shell formation and break down (uncoating) and chromatin tethering, all of which are required for all retroviral infections.

Retroviral Gag polyproteins are processed into at least three proteins, matrix (MA), capsid (CA) and nucleocapsid (NC), that form the structure of the virion

Figure 1: Schematic representation of retroviral Gag proteins. All retroviruses encode three main genes: gag, pol and env. The gag gene codes for the structural proteins of the virus. It is translated into a polyprotein that is then cleaved during viral maturation in orthoretroviruses into individual proteins as indicated in the key: matrix (MA, green); capsid (CA, red); nucleocapsid (NC, blue) and additional small proteins (purple and pink). The Gag protein of spumaretroviruses is not cleaved, shown in brown. The position of proline-rich late domain motifs which recruit the ESCRT pathway to facilitate viral release in late replication are indicated in yellow. The identified chromatin binding motifs are shown in light blue. The p12 amino acid sequence from the gammaretrovirus Mo-MLV is given, highlighting the motif in the NTD responsible for CA binding (in purple) and the CTD chromatin binding motif (in light blue). Abbreviations: PFV, prototypic foamy virus; HIV, human inmmunodeficiency virus; BLV, bovine leukemia virus; WDSV, walleye dermal sarcoma virus; RSV, Rous sarcoma virus; M-PMV, Mason Pfizer Monkey virus; MLV, murine leukemia virus.

Bishop lab p12 diagram

Figure 2. A model for gammaretroviral p12 function in the early stages of infection. In the mature virion, the NTD of p12 directly binds to and stabilises the hexameric CA lattice that surrounds the viral core. The gammaretroviral PIC, minimally carrying p12, CA, IN and the viral cDNA must wait until mitosis, when the nuclear envelope disassembles, to gain access to host chromatin. The PIC is then tethered to nucleosomes by CA-bound, phosphorylated p12. Exit from mitosis triggers the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. This then reveals the intasome, consisting of viral cDNA and IN. IN can then bind to chromatin-associated BET proteins and direct the viral cDNA to gene promoter regions where it then catalyses integration of the viral cDNA into host chromatin to form a provirus.