HIV-1 exploits numerous host cellular pathways for productive
infection. To identify novel factors involved in HIV-1 replication,
HIV-1 integrase and matrix
protein complexes were captured at 4 hours post
infection for proteomic analysis using an affinity purification system.
Leucine-rich PPR-motif containing (LRPPRC)
protein, a cellular
protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1
nucleic acids during the early steps of
virus infection. To establish if LRPPRC was critical for HIV-1
infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1
infection was reduced in all three cell lines, but virus production and
RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus
infection, a virus dependent on cellular proliferation for productive
infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of
virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but
viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more than one mechanism.