The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration.
Author Summary All retroviruses reverse transcribe their RNA genome to a DNA copy in the cytoplasm of the infected cell. To be expressed, the viral genomic DNA has to travel to the cell nucleus and to integrate into the cellular chromosomes. This trafficking is governed by cellular and viral proteins that associate with the viral genome to form a ‘pre-integration complex’ (PIC), yet the full composition of this complex is unknown. Former studies showed that for the murine leukemia virus (MLV), mutations in a viral protein named p12 abrogate MLV infection, after reverse transcription and prior to the integration step, suggesting a role for this protein in early stages of infection. However, the precise mechanism of p12 action is not known. We combined microscopic, genetic and biochemical techniques to provide evidence that the p12 protein is part of the MLV PIC and that it exerts its function from within this complex. These analyses also suggest a role for p12 in the trafficking of the PIC from the cytoplasm to the chromosomes of the infected cell. Altogether, these findings highlight an important ‘building block’ of a complex that is essential for MLV infection.