Abstract
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.
Highlights
Efficient replication of human immunodeficiency virus type 1 (HIV-1) requires interactions with a myriad of host cell proteins
Identification of Leucine-rich pentatricopeptide repeat (PPR)-motif containing (LRPPRC) To investigate the proteome of HIV-1 protein complexes, we utilized a previously described biotinylation system to affinity capture HIV-1 integrase (IN) and matrix (MA) protein complexes in vivo [38]
Retroviral replication is a complex interaction between virus and host factors. These studies identified a new cellular protein, LRPPRC, which is necessary for efficient HIV-1 infection
Summary
Efficient replication of human immunodeficiency virus type 1 (HIV-1) requires interactions with a myriad of host cell proteins. After entry and uncoating of its viral core, there are many critical steps during HIV-1 replication, including, but not limited to, reverse transcription of the viral RNA (vRNA) into DNA, nuclear import of the viral DNA (vDNA), and the integration of the vDNA into the host cell chromosome, transcription, specific export of unspliced viral mRNA, assembly of new virus particles, virion egress, and maturation. All of these steps involve a complex interplay between viral and cellular proteins [2,3]. MA is a component of both reverse transcription and preintegration complexes and contains two putative nuclear localization sequences (NLS) [10]; deletion of these sequences does not ablate the nuclear import process [11,12,13]
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