Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.

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.

In vivo biotinylation and capture of HIV-1 matrix and integrase proteins.

This report describes the adaptation of the biotin ligase BirA-biotin acceptor sequence (BAS) labeling system to biotinylate specific human immunodeficiency virus 1 (HIV-1) proteins in vivo. Two HIV-1 clones were constructed, with the BAS introduced into the matrix region of gag or the integrase region of pol. Specific biotinylation of target proteins in virions was observed when molecular clones were co-expressed with BirA. Both BAS-containing viruses propagated in SupT1 T-cells although replication of the integrase clone was delayed. Further studies demonstrated that the integrase insertion yielded an approximate 40% reduction in single-round infectivity as assessed on MAGI-5 indicator cells, as well as in the in vitro integration activity of preintegration complexes extracted from acutely infected C8166-45 T-cells. Biotinylation of the integrase BAS tag furthermore rendered this virus non-infectious. The matrix viral clone by contrast displayed wild-type behavior under all conditions tested. These results therefore establish a system whereby biotinylated matrix protein in the context of replication-competent virus could be used to label and capture viral protein complexes in vivo.