Abstract
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.
Highlights
Reverse transcription and integration are the hallmarks of the retroviral life cycle
Reverse transcription occurs in a cytoplasmic complex, termed reverse transcription complex (RTC), which transforms to the pre-integration complex (PIC)
Genetic and biochemical techniques to provide evidence that the p12 protein is part of the murine leukemia virus (MLV) PIC and that it exerts its function from within this complex. These analyses suggest a role for p12 in the trafficking of the PIC from the cytoplasm to the chromosomes of the infected cell
Summary
Reverse transcription and integration are the hallmarks of the retroviral life cycle These steps include reverse transcription of the genomic RNA into a linear double-stranded DNA and the subsequent integration of this DNA into the genome of the infected cell. These events are part of the ‘early’ stages of the retroviral life cycle, starting with the binding of the virus to its cellular receptor and ending once the integration step has occurred. Reverse transcription occurs in a cytoplasmic complex, termed reverse transcription complex (RTC), which transforms to the PIC (reviewed in [1,2]). The PIC harbors the viral DNA and travels from the cytoplasm to the nucleus, to target the chromatin of the infected cell for integration
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